SRX5619605 GSM3699823 SRP190015 SRS4563286 GSM3699823 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699823 GSM3699823 GEO Accession GSM3699823 SRX5619606 GSM3699824 SRP190015 SRS4563287 GSM3699824 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699824 GSM3699824 GEO Accession GSM3699824 SRX5619607 GSM3699825 SRP190015 SRS4563288 GSM3699825 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699825 GSM3699825 GEO Accession GSM3699825 SRX5619608 GSM3699826 SRP190015 SRS4563289 GSM3699826 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699826 GSM3699826 GEO Accession GSM3699826 SRX5619609 GSM3699827 SRP190015 SRS4563290 GSM3699827 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699827 GSM3699827 GEO Accession GSM3699827 SRX5619610 GSM3699828 SRP190015 SRS4563292 GSM3699828 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699828 GSM3699828 GEO Accession GSM3699828 SRX5619611 GSM3699829 SRP190015 SRS4563293 GSM3699829 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699829 GSM3699829 GEO Accession GSM3699829 SRX5619576 GSM3699794 SRP190015 SRS4563259 GSM3699794 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699794 GSM3699794 GEO Accession GSM3699794 SRX5619577 GSM3699795 SRP190015 SRS4563258 GSM3699795 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699795 GSM3699795 GEO Accession GSM3699795 SRX5619578 GSM3699796 SRP190015 SRS4563257 GSM3699796 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699796 GSM3699796 GEO Accession GSM3699796 SRX5619579 GSM3699797 SRP190015 SRS4563260 GSM3699797 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699797 GSM3699797 GEO Accession GSM3699797 SRX5619580 GSM3699798 SRP190015 SRS4563261 GSM3699798 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699798 GSM3699798 GEO Accession GSM3699798 SRX5619581 GSM3699799 SRP190015 SRS4563262 GSM3699799 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699799 GSM3699799 GEO Accession GSM3699799 SRX5619582 GSM3699800 SRP190015 SRS4563263 GSM3699800 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699800 GSM3699800 GEO Accession GSM3699800 SRX5619583 GSM3699801 SRP190015 SRS4563264 GSM3699801 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699801 GSM3699801 GEO Accession GSM3699801 SRX5619584 GSM3699802 SRP190015 SRS4563265 GSM3699802 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699802 GSM3699802 GEO Accession GSM3699802 SRX5619585 GSM3699803 SRP190015 SRS4563266 GSM3699803 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699803 GSM3699803 GEO Accession GSM3699803 SRX5619586 GSM3699804 SRP190015 SRS4563267 GSM3699804 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699804 GSM3699804 GEO Accession GSM3699804 SRX5619587 GSM3699805 SRP190015 SRS4563269 GSM3699805 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699805 GSM3699805 GEO Accession GSM3699805 SRX5619588 GSM3699806 SRP190015 SRS4563268 GSM3699806 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699806 GSM3699806 GEO Accession GSM3699806 SRX5619589 GSM3699807 SRP190015 SRS4563270 GSM3699807 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699807 GSM3699807 GEO Accession GSM3699807 SRX5619590 GSM3699808 SRP190015 SRS4563271 GSM3699808 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699808 GSM3699808 GEO Accession GSM3699808 SRX5619591 GSM3699809 SRP190015 SRS4563272 GSM3699809 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699809 GSM3699809 GEO Accession GSM3699809 SRX5619592 GSM3699810 SRP190015 SRS4563273 GSM3699810 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699810 GSM3699810 GEO Accession GSM3699810 SRX5619593 GSM3699811 SRP190015 SRS4563274 GSM3699811 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699811 GSM3699811 GEO Accession GSM3699811 SRX5619594 GSM3699812 SRP190015 SRS4563276 GSM3699812 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699812 GSM3699812 GEO Accession GSM3699812 SRX5619595 GSM3699813 SRP190015 SRS4563275 GSM3699813 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699813 GSM3699813 GEO Accession GSM3699813 SRX5619596 GSM3699814 SRP190015 SRS4563277 GSM3699814 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699814 GSM3699814 GEO Accession GSM3699814 SRX5619597 GSM3699815 SRP190015 SRS4563279 GSM3699815 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699815 GSM3699815 GEO Accession GSM3699815 SRX5619598 GSM3699816 SRP190015 SRS4563278 GSM3699816 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699816 GSM3699816 GEO Accession GSM3699816 SRX5619599 GSM3699817 SRP190015 SRS4563280 GSM3699817 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699817 GSM3699817 GEO Accession GSM3699817 SRX5619600 GSM3699818 SRP190015 SRS4563281 GSM3699818 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699818 GSM3699818 GEO Accession GSM3699818 SRX5619601 GSM3699819 SRP190015 SRS4563282 GSM3699819 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699819 GSM3699819 GEO Accession GSM3699819 SRX5619602 GSM3699820 SRP190015 SRS4563283 GSM3699820 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699820 GSM3699820 GEO Accession GSM3699820 SRX5619603 GSM3699821 SRP190015 SRS4563285 GSM3699821 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699821 GSM3699821 GEO Accession GSM3699821 SRX5619604 GSM3699822 SRP190015 SRS4563284 GSM3699822 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699822 GSM3699822 GEO Accession GSM3699822 SRX5619612 GSM3699830 SRP190015 SRS4563291 GSM3699830 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699830 GSM3699830 GEO Accession GSM3699830 SRX5619613 GSM3699831 SRP190015 SRS4563294 GSM3699831 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699831 GSM3699831 GEO Accession GSM3699831 SRX5619614 GSM3699832 SRP190015 SRS4563297 GSM3699832 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699832 GSM3699832 GEO Accession GSM3699832 SRX5619615 GSM3699833 SRP190015 SRS4563295 GSM3699833 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699833 GSM3699833 GEO Accession GSM3699833 SRX5619616 GSM3699834 SRP190015 SRS4563296 GSM3699834 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699834 GSM3699834 GEO Accession GSM3699834 SRX5619617 GSM3699835 SRP190015 SRS4563298 GSM3699835 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699835 GSM3699835 GEO Accession GSM3699835 SRX5619618 GSM3699836 SRP190015 SRS4563300 GSM3699836 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699836 GSM3699836 GEO Accession GSM3699836 SRX5619619 GSM3699837 SRP190015 SRS4563299 GSM3699837 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699837 GSM3699837 GEO Accession GSM3699837 SRX5619620 GSM3699838 SRP190015 SRS4563301 GSM3699838 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699838 GSM3699838 GEO Accession GSM3699838 SRX5619621 GSM3699839 SRP190015 SRS4563302 GSM3699839 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699839 GSM3699839 GEO Accession GSM3699839 SRX5619622 GSM3699840 SRP190015 SRS4563303 GSM3699840 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699840 GSM3699840 GEO Accession GSM3699840 SRX5619623 GSM3699841 SRP190015 SRS4563304 GSM3699841 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699841 GSM3699841 GEO Accession GSM3699841 SRX5619624 GSM3699842 SRP190015 SRS4563305 GSM3699842 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699842 GSM3699842 GEO Accession GSM3699842 SRX5619625 GSM3699843 SRP190015 SRS4563306 GSM3699843 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699843 GSM3699843 GEO Accession GSM3699843 SRX5619626 GSM3699844 SRP190015 SRS4563307 GSM3699844 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699844 GSM3699844 GEO Accession GSM3699844 SRX5619627 GSM3699845 SRP190015 SRS4563309 GSM3699845 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699845 GSM3699845 GEO Accession GSM3699845 SRX5619628 GSM3699846 SRP190015 SRS4563308 GSM3699846 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699846 GSM3699846 GEO Accession GSM3699846 SRX5619629 GSM3699847 SRP190015 SRS4563310 GSM3699847 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699847 GSM3699847 GEO Accession GSM3699847 SRX5619630 GSM3699848 SRP190015 SRS4563311 GSM3699848 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699848 GSM3699848 GEO Accession GSM3699848 SRX5619631 GSM3699849 SRP190015 SRS4563313 GSM3699849 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699849 GSM3699849 GEO Accession GSM3699849 SRX5619632 GSM3699850 SRP190015 SRS4563312 GSM3699850 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699850 GSM3699850 GEO Accession GSM3699850 SRX5619633 GSM3699851 SRP190015 SRS4563314 GSM3699851 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699851 GSM3699851 GEO Accession GSM3699851 SRX5619634 GSM3699852 SRP190015 SRS4563315 GSM3699852 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699852 GSM3699852 GEO Accession GSM3699852 SRX5619635 GSM3699853 SRP190015 SRS4563316 GSM3699853 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699853 GSM3699853 GEO Accession GSM3699853 SRX5619636 GSM3699854 SRP190015 SRS4563317 GSM3699854 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699854 GSM3699854 GEO Accession GSM3699854 SRX5619637 GSM3699855 SRP190015 SRS4563318 GSM3699855 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699855 GSM3699855 GEO Accession GSM3699855 SRX5619638 GSM3699856 SRP190015 SRS4563319 GSM3699856 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699856 GSM3699856 GEO Accession GSM3699856 SRX5619639 GSM3699857 SRP190015 SRS4563320 GSM3699857 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699857 GSM3699857 GEO Accession GSM3699857 SRX5619640 GSM3699858 SRP190015 SRS4563322 GSM3699858 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699858 GSM3699858 GEO Accession GSM3699858 SRX5619641 GSM3699859 SRP190015 SRS4563321 GSM3699859 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699859 GSM3699859 GEO Accession GSM3699859 SRX5619642 GSM3699860 SRP190015 SRS4563323 GSM3699860 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699860 GSM3699860 GEO Accession GSM3699860 SRX5619643 GSM3699861 SRP190015 SRS4563324 GSM3699861 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699861 GSM3699861 GEO Accession GSM3699861 SRX5619644 GSM3699862 SRP190015 SRS4563325 GSM3699862 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699862 GSM3699862 GEO Accession GSM3699862 SRX5619645 GSM3699863 SRP190015 SRS4563326 GSM3699863 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699863 GSM3699863 GEO Accession GSM3699863 SRX5619646 GSM3699864 SRP190015 SRS4563329 GSM3699864 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699864 GSM3699864 GEO Accession GSM3699864 SRX5619647 GSM3699865 SRP190015 SRS4563327 GSM3699865 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699865 GSM3699865 GEO Accession GSM3699865 SRX5619648 GSM3699866 SRP190015 SRS4563328 GSM3699866 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699866 GSM3699866 GEO Accession GSM3699866 SRX5619649 GSM3699867 SRP190015 SRS4563330 GSM3699867 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699867 GSM3699867 GEO Accession GSM3699867 SRX5619650 GSM3699868 SRP190015 SRS4563331 GSM3699868 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699868 GSM3699868 GEO Accession GSM3699868 SRX5619651 GSM3699869 SRP190015 SRS4563332 GSM3699869 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699869 GSM3699869 GEO Accession GSM3699869 SRX5619652 GSM3699870 SRP190015 SRS4563334 GSM3699870 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699870 GSM3699870 GEO Accession GSM3699870 SRX5619653 GSM3699871 SRP190015 SRS4563333 GSM3699871 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699871 GSM3699871 GEO Accession GSM3699871 SRX5619654 GSM3699872 SRP190015 SRS4563335 GSM3699872 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699872 GSM3699872 GEO Accession GSM3699872 SRX5619655 GSM3699873 SRP190015 SRS4563336 GSM3699873 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699873 GSM3699873 GEO Accession GSM3699873 SRX5619656 GSM3699874 SRP190015 SRS4563337 GSM3699874 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699874 GSM3699874 GEO Accession GSM3699874 SRX5619657 GSM3699875 SRP190015 SRS4563338 GSM3699875 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699875 GSM3699875 GEO Accession GSM3699875 SRX5619658 GSM3699876 SRP190015 SRS4563339 GSM3699876 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699876 GSM3699876 GEO Accession GSM3699876 SRX5619659 GSM3699877 SRP190015 SRS4563340 GSM3699877 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699877 GSM3699877 GEO Accession GSM3699877 SRX5619660 GSM3699878 SRP190015 SRS4563341 GSM3699878 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699878 GSM3699878 GEO Accession GSM3699878 SRX5619661 GSM3699879 SRP190015 SRS4563342 GSM3699879 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699879 GSM3699879 GEO Accession GSM3699879 SRX5619662 GSM3699880 SRP190015 SRS4563345 GSM3699880 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699880 GSM3699880 GEO Accession GSM3699880 SRX5619663 GSM3699881 SRP190015 SRS4563344 GSM3699881 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699881 GSM3699881 GEO Accession GSM3699881 SRX5619664 GSM3699882 SRP190015 SRS4563343 GSM3699882 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699882 GSM3699882 GEO Accession GSM3699882 SRX5619665 GSM3699883 SRP190015 SRS4563346 GSM3699883 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699883 GSM3699883 GEO Accession GSM3699883 SRX5619666 GSM3699884 SRP190015 SRS4563347 GSM3699884 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699884 GSM3699884 GEO Accession GSM3699884 SRX5619667 GSM3699885 SRP190015 SRS4563348 GSM3699885 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699885 GSM3699885 GEO Accession GSM3699885 SRX5619668 GSM3699886 SRP190015 SRS4563349 GSM3699886 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699886 GSM3699886 GEO Accession GSM3699886 SRX5619669 GSM3699887 SRP190015 SRS4563350 GSM3699887 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699887 GSM3699887 GEO Accession GSM3699887 SRX5619670 GSM3699888 SRP190015 SRS4563351 GSM3699888 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699888 GSM3699888 GEO Accession GSM3699888 SRX5619671 GSM3699889 SRP190015 SRS4563352 GSM3699889 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699889 GSM3699889 GEO Accession GSM3699889 SRX5619672 GSM3699890 SRP190015 SRS4563353 GSM3699890 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699890 GSM3699890 GEO Accession GSM3699890 SRX5619673 GSM3699891 SRP190015 SRS4563355 GSM3699891 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699891 GSM3699891 GEO Accession GSM3699891 SRX5619674 GSM3699892 SRP190015 SRS4563354 GSM3699892 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699892 GSM3699892 GEO Accession GSM3699892 SRX5619675 GSM3699893 SRP190015 SRS4563356 GSM3699893 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699893 GSM3699893 GEO Accession GSM3699893 SRX5619676 GSM3699894 SRP190015 SRS4563357 GSM3699894 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699894 GSM3699894 GEO Accession GSM3699894 SRX5619677 GSM3699895 SRP190015 SRS4563358 GSM3699895 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699895 GSM3699895 GEO Accession GSM3699895 SRX5619678 GSM3699896 SRP190015 SRS4563359 GSM3699896 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699896 GSM3699896 GEO Accession GSM3699896 SRX5619679 GSM3699897 SRP190015 SRS4563360 GSM3699897 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699897 GSM3699897 GEO Accession GSM3699897 SRX5619680 GSM3699898 SRP190015 SRS4563361 GSM3699898 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699898 GSM3699898 GEO Accession GSM3699898 SRX5619681 GSM3699899 SRP190015 SRS4563362 GSM3699899 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699899 GSM3699899 GEO Accession GSM3699899 SRX5619682 GSM3699900 SRP190015 SRS4563363 GSM3699900 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699900 GSM3699900 GEO Accession GSM3699900 SRX5619683 GSM3699901 SRP190015 SRS4563365 GSM3699901 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699901 GSM3699901 GEO Accession GSM3699901 SRX5619684 GSM3699902 SRP190015 SRS4563366 GSM3699902 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699902 GSM3699902 GEO Accession GSM3699902 SRX5619685 GSM3699903 SRP190015 SRS4563364 GSM3699903 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699903 GSM3699903 GEO Accession GSM3699903 SRX5619686 GSM3699904 SRP190015 SRS4563367 GSM3699904 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699904 GSM3699904 GEO Accession GSM3699904 SRX5619687 GSM3699905 SRP190015 SRS4563368 GSM3699905 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699905 GSM3699905 GEO Accession GSM3699905 SRX5619688 GSM3699906 SRP190015 SRS4563370 GSM3699906 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699906 GSM3699906 GEO Accession GSM3699906 SRX5619689 GSM3699907 SRP190015 SRS4563369 GSM3699907 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699907 GSM3699907 GEO Accession GSM3699907 SRX5619690 GSM3699908 SRP190015 SRS4563371 GSM3699908 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699908 GSM3699908 GEO Accession GSM3699908 SRX5619691 GSM3699909 SRP190015 SRS4563373 GSM3699909 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699909 GSM3699909 GEO Accession GSM3699909 SRX5619692 GSM3699910 SRP190015 SRS4563372 GSM3699910 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699910 GSM3699910 GEO Accession GSM3699910 SRX5619693 GSM3699911 SRP190015 SRS4563374 GSM3699911 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699911 GSM3699911 GEO Accession GSM3699911 SRX5619694 GSM3699912 SRP190015 SRS4563375 GSM3699912 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699912 GSM3699912 GEO Accession GSM3699912 SRX5619695 GSM3699913 SRP190015 SRS4563376 GSM3699913 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699913 GSM3699913 GEO Accession GSM3699913 SRX5619696 GSM3699914 SRP190015 SRS4563377 GSM3699914 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699914 GSM3699914 GEO Accession GSM3699914 SRX5619697 GSM3699915 SRP190015 SRS4563378 GSM3699915 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699915 GSM3699915 GEO Accession GSM3699915 SRX5619698 GSM3699916 SRP190015 SRS4563379 GSM3699916 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699916 GSM3699916 GEO Accession GSM3699916 SRX5619699 GSM3699917 SRP190015 SRS4563380 GSM3699917 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699917 GSM3699917 GEO Accession GSM3699917 SRX5619700 GSM3699918 SRP190015 SRS4563382 GSM3699918 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699918 GSM3699918 GEO Accession GSM3699918 SRX5619701 GSM3699919 SRP190015 SRS4563381 GSM3699919 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699919 GSM3699919 GEO Accession GSM3699919 SRX5619702 GSM3699920 SRP190015 SRS4563383 GSM3699920 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699920 GSM3699920 GEO Accession GSM3699920 SRX5619703 GSM3699921 SRP190015 SRS4563384 GSM3699921 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699921 GSM3699921 GEO Accession GSM3699921 SRX5619704 GSM3699922 SRP190015 SRS4563385 GSM3699922 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699922 GSM3699922 GEO Accession GSM3699922 SRX5619705 GSM3699923 SRP190015 SRS4563386 GSM3699923 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699923 GSM3699923 GEO Accession GSM3699923 SRX5619706 GSM3699924 SRP190015 SRS4563387 GSM3699924 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699924 GSM3699924 GEO Accession GSM3699924 SRX5619707 GSM3699925 SRP190015 SRS4563388 GSM3699925 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699925 GSM3699925 GEO Accession GSM3699925 SRX5619708 GSM3699926 SRP190015 SRS4563390 GSM3699926 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699926 GSM3699926 GEO Accession GSM3699926 SRX5619709 GSM3699927 SRP190015 SRS4563389 GSM3699927 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699927 GSM3699927 GEO Accession GSM3699927 SRX5619710 GSM3699928 SRP190015 SRS4563391 GSM3699928 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699928 GSM3699928 GEO Accession GSM3699928 SRX5619711 GSM3699929 SRP190015 SRS4563392 GSM3699929 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699929 GSM3699929 GEO Accession GSM3699929 SRX5619712 GSM3699930 SRP190015 SRS4563393 GSM3699930 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699930 GSM3699930 GEO Accession GSM3699930 SRX5619713 GSM3699931 SRP190015 SRS4563394 GSM3699931 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699931 GSM3699931 GEO Accession GSM3699931 SRX5619714 GSM3699932 SRP190015 SRS4563396 GSM3699932 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699932 GSM3699932 GEO Accession GSM3699932 SRX5619715 GSM3699933 SRP190015 SRS4563395 GSM3699933 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699933 GSM3699933 GEO Accession GSM3699933 SRX5619716 GSM3699934 SRP190015 SRS4563397 GSM3699934 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699934 GSM3699934 GEO Accession GSM3699934 SRX5619717 GSM3699935 SRP190015 SRS4563398 GSM3699935 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699935 GSM3699935 GEO Accession GSM3699935 SRX5619718 GSM3699936 SRP190015 SRS4563399 GSM3699936 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699936 GSM3699936 GEO Accession GSM3699936 SRX5619719 GSM3699937 SRP190015 SRS4563400 GSM3699937 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699937 GSM3699937 GEO Accession GSM3699937 SRX5619720 GSM3699938 SRP190015 SRS4563402 GSM3699938 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699938 GSM3699938 GEO Accession GSM3699938 SRX5619721 GSM3699939 SRP190015 SRS4563401 GSM3699939 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699939 GSM3699939 GEO Accession GSM3699939 SRX5619722 GSM3699940 SRP190015 SRS4563403 GSM3699940 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699940 GSM3699940 GEO Accession GSM3699940 SRX5619723 GSM3699941 SRP190015 SRS4563404 GSM3699941 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699941 GSM3699941 GEO Accession GSM3699941 SRX5619724 GSM3699942 SRP190015 SRS4563407 GSM3699942 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699942 GSM3699942 GEO Accession GSM3699942 SRX5619725 GSM3699943 SRP190015 SRS4563406 GSM3699943 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699943 GSM3699943 GEO Accession GSM3699943 SRX5619726 GSM3699944 SRP190015 SRS4563405 GSM3699944 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699944 GSM3699944 GEO Accession GSM3699944 SRX5619727 GSM3699945 SRP190015 SRS4563408 GSM3699945 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699945 GSM3699945 GEO Accession GSM3699945 SRX5619728 GSM3699946 SRP190015 SRS4563409 GSM3699946 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699946 GSM3699946 GEO Accession GSM3699946 SRX5619729 GSM3699947 SRP190015 SRS4563410 GSM3699947 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699947 GSM3699947 GEO Accession GSM3699947 SRX5619730 GSM3699948 SRP190015 SRS4563411 GSM3699948 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699948 GSM3699948 GEO Accession GSM3699948 SRX5619731 GSM3699949 SRP190015 SRS4563412 GSM3699949 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699949 GSM3699949 GEO Accession GSM3699949 SRX5619732 GSM3699950 SRP190015 SRS4563413 GSM3699950 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699950 GSM3699950 GEO Accession GSM3699950 SRX5619733 GSM3699951 SRP190015 SRS4563414 GSM3699951 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699951 GSM3699951 GEO Accession GSM3699951 SRX5619734 GSM3699952 SRP190015 SRS4563415 GSM3699952 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699952 GSM3699952 GEO Accession GSM3699952 SRX5619735 GSM3699953 SRP190015 SRS4563416 GSM3699953 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699953 GSM3699953 GEO Accession GSM3699953 SRX5619736 GSM3699954 SRP190015 SRS4563417 GSM3699954 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699954 GSM3699954 GEO Accession GSM3699954 SRX5619737 GSM3699955 SRP190015 SRS4563418 GSM3699955 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699955 GSM3699955 GEO Accession GSM3699955 SRX5619738 GSM3699956 SRP190015 SRS4563419 GSM3699956 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699956 GSM3699956 GEO Accession GSM3699956 SRX5619739 GSM3699957 SRP190015 SRS4563421 GSM3699957 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699957 GSM3699957 GEO Accession GSM3699957 SRX5619740 GSM3699958 SRP190015 SRS4563420 GSM3699958 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699958 GSM3699958 GEO Accession GSM3699958 SRX5619741 GSM3699959 SRP190015 SRS4563422 GSM3699959 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699959 GSM3699959 GEO Accession GSM3699959 SRX5619742 GSM3699960 SRP190015 SRS4563424 GSM3699960 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699960 GSM3699960 GEO Accession GSM3699960 SRX5619743 GSM3699961 SRP190015 SRS4563423 GSM3699961 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699961 GSM3699961 GEO Accession GSM3699961 SRX5619744 GSM3699962 SRP190015 SRS4563425 GSM3699962 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699962 GSM3699962 GEO Accession GSM3699962 SRX5619745 GSM3699963 SRP190015 SRS4563426 GSM3699963 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699963 GSM3699963 GEO Accession GSM3699963 SRX5619746 GSM3699964 SRP190015 SRS4563427 GSM3699964 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699964 GSM3699964 GEO Accession GSM3699964 SRX5619747 GSM3699965 SRP190015 SRS4563428 GSM3699965 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699965 GSM3699965 GEO Accession GSM3699965 SRX5619748 GSM3699966 SRP190015 SRS4563431 GSM3699966 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699966 GSM3699966 GEO Accession GSM3699966 SRX5619749 GSM3699967 SRP190015 SRS4563430 GSM3699967 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699967 GSM3699967 GEO Accession GSM3699967 SRX5619750 GSM3699968 SRP190015 SRS4563429 GSM3699968 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699968 GSM3699968 GEO Accession GSM3699968 SRX5619751 GSM3699969 SRP190015 SRS4563432 GSM3699969 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699969 GSM3699969 GEO Accession GSM3699969 SRX5619752 GSM3699970 SRP190015 SRS4563433 GSM3699970 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699970 GSM3699970 GEO Accession GSM3699970 SRX5619753 GSM3699971 SRP190015 SRS4563434 GSM3699971 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699971 GSM3699971 GEO Accession GSM3699971 SRX5619754 GSM3699972 SRP190015 SRS4563436 GSM3699972 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699972 GSM3699972 GEO Accession GSM3699972 SRX5619755 GSM3699973 SRP190015 SRS4563435 GSM3699973 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699973 GSM3699973 GEO Accession GSM3699973 SRX5619756 GSM3699974 SRP190015 SRS4563437 GSM3699974 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699974 GSM3699974 GEO Accession GSM3699974 SRX5619757 GSM3699975 SRP190015 SRS4563438 GSM3699975 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699975 GSM3699975 GEO Accession GSM3699975 SRX5619758 GSM3699976 SRP190015 SRS4563439 GSM3699976 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699976 GSM3699976 GEO Accession GSM3699976 SRX5619759 GSM3699977 SRP190015 SRS4563440 GSM3699977 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699977 GSM3699977 GEO Accession GSM3699977 SRX5619760 GSM3699978 SRP190015 SRS4563441 GSM3699978 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699978 GSM3699978 GEO Accession GSM3699978 SRX5619761 GSM3699979 SRP190015 SRS4563444 GSM3699979 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699979 GSM3699979 GEO Accession GSM3699979 SRX5619762 GSM3699980 SRP190015 SRS4563442 GSM3699980 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699980 GSM3699980 GEO Accession GSM3699980 SRX5619763 GSM3699981 SRP190015 SRS4563443 GSM3699981 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699981 GSM3699981 GEO Accession GSM3699981 SRX5619764 GSM3699982 SRP190015 SRS4563445 GSM3699982 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699982 GSM3699982 GEO Accession GSM3699982 SRX5619765 GSM3699983 SRP190015 SRS4563446 GSM3699983 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699983 GSM3699983 GEO Accession GSM3699983 SRX5619766 GSM3699984 SRP190015 SRS4563447 GSM3699984 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699984 GSM3699984 GEO Accession GSM3699984 SRX5619767 GSM3699985 SRP190015 SRS4563448 GSM3699985 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699985 GSM3699985 GEO Accession GSM3699985 SRX5619768 GSM3699986 SRP190015 SRS4563449 GSM3699986 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699986 GSM3699986 GEO Accession GSM3699986 SRX5619769 GSM3699987 SRP190015 SRS4563450 GSM3699987 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699987 GSM3699987 GEO Accession GSM3699987 SRX5619770 GSM3699988 SRP190015 SRS4563451 GSM3699988 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699988 GSM3699988 GEO Accession GSM3699988 SRX5619771 GSM3699989 SRP190015 SRS4563452 GSM3699989 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699989 GSM3699989 GEO Accession GSM3699989 SRX5619772 GSM3699990 SRP190015 SRS4563453 GSM3699990 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699990 GSM3699990 GEO Accession GSM3699990 SRX5619773 GSM3699991 SRP190015 SRS4563454 GSM3699991 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699991 GSM3699991 GEO Accession GSM3699991 SRX5619774 GSM3699992 SRP190015 SRS4563455 GSM3699992 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699992 GSM3699992 GEO Accession GSM3699992 SRX5619775 GSM3699993 SRP190015 SRS4563457 GSM3699993 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699993 GSM3699993 GEO Accession GSM3699993 SRX5619776 GSM3699994 SRP190015 SRS4563456 GSM3699994 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699994 GSM3699994 GEO Accession GSM3699994 SRX5619777 GSM3699995 SRP190015 SRS4563458 GSM3699995 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699995 GSM3699995 GEO Accession GSM3699995 SRX5619778 GSM3699996 SRP190015 SRS4563459 GSM3699996 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699996 GSM3699996 GEO Accession GSM3699996 SRX5619779 GSM3699997 SRP190015 SRS4563461 GSM3699997 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699997 GSM3699997 GEO Accession GSM3699997 SRX5619780 GSM3699998 SRP190015 SRS4563460 GSM3699998 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699998 GSM3699998 GEO Accession GSM3699998 SRX5619781 GSM3699999 SRP190015 SRS4563462 GSM3699999 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303699999 GSM3699999 GEO Accession GSM3699999 SRX5619782 GSM3700000 SRP190015 SRS4563463 GSM3700000 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700000 GSM3700000 GEO Accession GSM3700000 SRX5619783 GSM3700001 SRP190015 SRS4563465 GSM3700001 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700001 GSM3700001 GEO Accession GSM3700001 SRX5619784 GSM3700002 SRP190015 SRS4563466 GSM3700002 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700002 GSM3700002 GEO Accession GSM3700002 SRX5619785 GSM3700003 SRP190015 SRS4563464 GSM3700003 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700003 GSM3700003 GEO Accession GSM3700003 SRX5619786 GSM3700004 SRP190015 SRS4563468 GSM3700004 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700004 GSM3700004 GEO Accession GSM3700004 SRX5619787 GSM3700005 SRP190015 SRS4563467 GSM3700005 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700005 GSM3700005 GEO Accession GSM3700005 SRX5619788 GSM3700006 SRP190015 SRS4563470 GSM3700006 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700006 GSM3700006 GEO Accession GSM3700006 SRX5619789 GSM3700007 SRP190015 SRS4563469 GSM3700007 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700007 GSM3700007 GEO Accession GSM3700007 SRX5619790 GSM3700008 SRP190015 SRS4563471 GSM3700008 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700008 GSM3700008 GEO Accession GSM3700008 SRX5619791 GSM3700009 SRP190015 SRS4563472 GSM3700009 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700009 GSM3700009 GEO Accession GSM3700009 SRX5619792 GSM3700010 SRP190015 SRS4563474 GSM3700010 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700010 GSM3700010 GEO Accession GSM3700010 SRX5619793 GSM3700011 SRP190015 SRS4563473 GSM3700011 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700011 GSM3700011 GEO Accession GSM3700011 SRX5619794 GSM3700012 SRP190015 SRS4563475 GSM3700012 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700012 GSM3700012 GEO Accession GSM3700012 SRX5619795 GSM3700013 SRP190015 SRS4563476 GSM3700013 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700013 GSM3700013 GEO Accession GSM3700013 SRX5619796 GSM3700014 SRP190015 SRS4563479 GSM3700014 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700014 GSM3700014 GEO Accession GSM3700014 SRX5619797 GSM3700015 SRP190015 SRS4563478 GSM3700015 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700015 GSM3700015 GEO Accession GSM3700015 SRX5619798 GSM3700016 SRP190015 SRS4563477 GSM3700016 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700016 GSM3700016 GEO Accession GSM3700016 SRX5619799 GSM3700017 SRP190015 SRS4563480 GSM3700017 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700017 GSM3700017 GEO Accession GSM3700017 SRX5619800 GSM3700018 SRP190015 SRS4563481 GSM3700018 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700018 GSM3700018 GEO Accession GSM3700018 SRX5619801 GSM3700019 SRP190015 SRS4563482 GSM3700019 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700019 GSM3700019 GEO Accession GSM3700019 SRX5619802 GSM3700020 SRP190015 SRS4563483 GSM3700020 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700020 GSM3700020 GEO Accession GSM3700020 SRX5619803 GSM3700021 SRP190015 SRS4563485 GSM3700021 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700021 GSM3700021 GEO Accession GSM3700021 SRX5619804 GSM3700022 SRP190015 SRS4563484 GSM3700022 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700022 GSM3700022 GEO Accession GSM3700022 SRX5619805 GSM3700023 SRP190015 SRS4563487 GSM3700023 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700023 GSM3700023 GEO Accession GSM3700023 SRX5619806 GSM3700024 SRP190015 SRS4563486 GSM3700024 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700024 GSM3700024 GEO Accession GSM3700024 SRX5619807 GSM3700025 SRP190015 SRS4563488 GSM3700025 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700025 GSM3700025 GEO Accession GSM3700025 SRX5619808 GSM3700026 SRP190015 SRS4563489 GSM3700026 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700026 GSM3700026 GEO Accession GSM3700026 SRX5619809 GSM3700027 SRP190015 SRS4563491 GSM3700027 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700027 GSM3700027 GEO Accession GSM3700027 SRX5619810 GSM3700028 SRP190015 SRS4563490 GSM3700028 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700028 GSM3700028 GEO Accession GSM3700028 SRX5619811 GSM3700029 SRP190015 SRS4563492 GSM3700029 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700029 GSM3700029 GEO Accession GSM3700029 SRX5619812 GSM3700030 SRP190015 SRS4563493 GSM3700030 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700030 GSM3700030 GEO Accession GSM3700030 SRX5619813 GSM3700031 SRP190015 SRS4563495 GSM3700031 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700031 GSM3700031 GEO Accession GSM3700031 SRX5619814 GSM3700032 SRP190015 SRS4563496 GSM3700032 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700032 GSM3700032 GEO Accession GSM3700032 SRX5619815 GSM3700033 SRP190015 SRS4563494 GSM3700033 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700033 GSM3700033 GEO Accession GSM3700033 SRX5619816 GSM3700034 SRP190015 SRS4563497 GSM3700034 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700034 GSM3700034 GEO Accession GSM3700034 SRX5619817 GSM3700035 SRP190015 SRS4563498 GSM3700035 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700035 GSM3700035 GEO Accession GSM3700035 SRX5619818 GSM3700036 SRP190015 SRS4563501 GSM3700036 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700036 GSM3700036 GEO Accession GSM3700036 SRX5619819 GSM3700037 SRP190015 SRS4563499 GSM3700037 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700037 GSM3700037 GEO Accession GSM3700037 SRX5619820 GSM3700038 SRP190015 SRS4563500 GSM3700038 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700038 GSM3700038 GEO Accession GSM3700038 SRX5619821 GSM3700039 SRP190015 SRS4563502 GSM3700039 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700039 GSM3700039 GEO Accession GSM3700039 SRX5619822 GSM3700040 SRP190015 SRS4563503 GSM3700040 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700040 GSM3700040 GEO Accession GSM3700040 SRX5619823 GSM3700041 SRP190015 SRS4563504 GSM3700041 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700041 GSM3700041 GEO Accession GSM3700041 SRX5619824 GSM3700042 SRP190015 SRS4563505 GSM3700042 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700042 GSM3700042 GEO Accession GSM3700042 SRX5619825 GSM3700043 SRP190015 SRS4563506 GSM3700043 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700043 GSM3700043 GEO Accession GSM3700043 SRX5619826 GSM3700044 SRP190015 SRS4563507 GSM3700044 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700044 GSM3700044 GEO Accession GSM3700044 SRX5619827 GSM3700045 SRP190015 SRS4563508 GSM3700045 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700045 GSM3700045 GEO Accession GSM3700045 SRX5619828 GSM3700046 SRP190015 SRS4563509 GSM3700046 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700046 GSM3700046 GEO Accession GSM3700046 SRX5619829 GSM3700047 SRP190015 SRS4563510 GSM3700047 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700047 GSM3700047 GEO Accession GSM3700047 SRX5619830 GSM3700048 SRP190015 SRS4563511 GSM3700048 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700048 GSM3700048 GEO Accession GSM3700048 SRX5619831 GSM3700049 SRP190015 SRS4563512 GSM3700049 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700049 GSM3700049 GEO Accession GSM3700049 SRX5619832 GSM3700050 SRP190015 SRS4563513 GSM3700050 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700050 GSM3700050 GEO Accession GSM3700050 SRX5619833 GSM3700051 SRP190015 SRS4563514 GSM3700051 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700051 GSM3700051 GEO Accession GSM3700051 SRX5619834 GSM3700052 SRP190015 SRS4563515 GSM3700052 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700052 GSM3700052 GEO Accession GSM3700052 SRX5619835 GSM3700053 SRP190015 SRS4563516 GSM3700053 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700053 GSM3700053 GEO Accession GSM3700053 SRX5619836 GSM3700054 SRP190015 SRS4563518 GSM3700054 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700054 GSM3700054 GEO Accession GSM3700054 SRX5619837 GSM3700055 SRP190015 SRS4563517 GSM3700055 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700055 GSM3700055 GEO Accession GSM3700055 SRX5619838 GSM3700056 SRP190015 SRS4563519 GSM3700056 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700056 GSM3700056 GEO Accession GSM3700056 SRX5619839 GSM3700057 SRP190015 SRS4563520 GSM3700057 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700057 GSM3700057 GEO Accession GSM3700057 SRX5619840 GSM3700058 SRP190015 SRS4563521 GSM3700058 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700058 GSM3700058 GEO Accession GSM3700058 SRX5619841 GSM3700059 SRP190015 SRS4563522 GSM3700059 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700059 GSM3700059 GEO Accession GSM3700059 SRX5619842 GSM3700060 SRP190015 SRS4563525 GSM3700060 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700060 GSM3700060 GEO Accession GSM3700060 SRX5619843 GSM3700061 SRP190015 SRS4563523 GSM3700061 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700061 GSM3700061 GEO Accession GSM3700061 SRX5619844 GSM3700062 SRP190015 SRS4563524 GSM3700062 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700062 GSM3700062 GEO Accession GSM3700062 SRX5619845 GSM3700063 SRP190015 SRS4563526 GSM3700063 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700063 GSM3700063 GEO Accession GSM3700063 SRX5619846 GSM3700064 SRP190015 SRS4563528 GSM3700064 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700064 GSM3700064 GEO Accession GSM3700064 SRX5619847 GSM3700065 SRP190015 SRS4563527 GSM3700065 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700065 GSM3700065 GEO Accession GSM3700065 SRX5619848 GSM3700066 SRP190015 SRS4563529 GSM3700066 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700066 GSM3700066 GEO Accession GSM3700066 SRX5619849 GSM3700067 SRP190015 SRS4563530 GSM3700067 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700067 GSM3700067 GEO Accession GSM3700067 SRX5619850 GSM3700068 SRP190015 SRS4563531 GSM3700068 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700068 GSM3700068 GEO Accession GSM3700068 SRX5619851 GSM3700069 SRP190015 SRS4563532 GSM3700069 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700069 GSM3700069 GEO Accession GSM3700069 SRX5619852 GSM3700070 SRP190015 SRS4563533 GSM3700070 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700070 GSM3700070 GEO Accession GSM3700070 SRX5619853 GSM3700071 SRP190015 SRS4563535 GSM3700071 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700071 GSM3700071 GEO Accession GSM3700071 SRX5619854 GSM3700072 SRP190015 SRS4563534 GSM3700072 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700072 GSM3700072 GEO Accession GSM3700072 SRX5619855 GSM3700073 SRP190015 SRS4563536 GSM3700073 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700073 GSM3700073 GEO Accession GSM3700073 SRX5619856 GSM3700074 SRP190015 SRS4563537 GSM3700074 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700074 GSM3700074 GEO Accession GSM3700074 SRX5619857 GSM3700075 SRP190015 SRS4563538 GSM3700075 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700075 GSM3700075 GEO Accession GSM3700075 SRX5619858 GSM3700076 SRP190015 SRS4563540 GSM3700076 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700076 GSM3700076 GEO Accession GSM3700076 SRX5619859 GSM3700077 SRP190015 SRS4563539 GSM3700077 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700077 GSM3700077 GEO Accession GSM3700077 SRX5619860 GSM3700078 SRP190015 SRS4563542 GSM3700078 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700078 GSM3700078 GEO Accession GSM3700078 SRX5619861 GSM3700079 SRP190015 SRS4563541 GSM3700079 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700079 GSM3700079 GEO Accession GSM3700079 SRX5619862 GSM3700080 SRP190015 SRS4563543 GSM3700080 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700080 GSM3700080 GEO Accession GSM3700080 SRX5619863 GSM3700081 SRP190015 SRS4563544 GSM3700081 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700081 GSM3700081 GEO Accession GSM3700081 SRX5619864 GSM3700082 SRP190015 SRS4563545 GSM3700082 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700082 GSM3700082 GEO Accession GSM3700082 SRX5619865 GSM3700083 SRP190015 SRS4563546 GSM3700083 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700083 GSM3700083 GEO Accession GSM3700083 SRX5619866 GSM3700084 SRP190015 SRS4563547 GSM3700084 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700084 GSM3700084 GEO Accession GSM3700084 SRX5619867 GSM3700085 SRP190015 SRS4563548 GSM3700085 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700085 GSM3700085 GEO Accession GSM3700085 SRX5619868 GSM3700086 SRP190015 SRS4563549 GSM3700086 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700086 GSM3700086 GEO Accession GSM3700086 SRX5619869 GSM3700087 SRP190015 SRS4563550 GSM3700087 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700087 GSM3700087 GEO Accession GSM3700087 SRX5619870 GSM3700088 SRP190015 SRS4563551 GSM3700088 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700088 GSM3700088 GEO Accession GSM3700088 SRX5619871 GSM3700089 SRP190015 SRS4563552 GSM3700089 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700089 GSM3700089 GEO Accession GSM3700089 SRX5619872 GSM3700090 SRP190015 SRS4563553 GSM3700090 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700090 GSM3700090 GEO Accession GSM3700090 SRX5619873 GSM3700091 SRP190015 SRS4563554 GSM3700091 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700091 GSM3700091 GEO Accession GSM3700091 SRX5619874 GSM3700092 SRP190015 SRS4563555 GSM3700092 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700092 GSM3700092 GEO Accession GSM3700092 SRX5619875 GSM3700093 SRP190015 SRS4563557 GSM3700093 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700093 GSM3700093 GEO Accession GSM3700093 SRX5619876 GSM3700094 SRP190015 SRS4563556 GSM3700094 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700094 GSM3700094 GEO Accession GSM3700094 SRX5619877 GSM3700095 SRP190015 SRS4563558 GSM3700095 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700095 GSM3700095 GEO Accession GSM3700095 SRX5619878 GSM3700096 SRP190015 SRS4563559 GSM3700096 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700096 GSM3700096 GEO Accession GSM3700096 SRX5619879 GSM3700097 SRP190015 SRS4563561 GSM3700097 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700097 GSM3700097 GEO Accession GSM3700097 SRX5619880 GSM3700098 SRP190015 SRS4563560 GSM3700098 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700098 GSM3700098 GEO Accession GSM3700098 SRX5619881 GSM3700099 SRP190015 SRS4563562 GSM3700099 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700099 GSM3700099 GEO Accession GSM3700099 SRX5619882 GSM3700100 SRP190015 SRS4563565 GSM3700100 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700100 GSM3700100 GEO Accession GSM3700100 SRX5619883 GSM3700101 SRP190015 SRS4563563 GSM3700101 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700101 GSM3700101 GEO Accession GSM3700101 SRX5619884 GSM3700102 SRP190015 SRS4563564 GSM3700102 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700102 GSM3700102 GEO Accession GSM3700102 SRX5619885 GSM3700103 SRP190015 SRS4563566 GSM3700103 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700103 GSM3700103 GEO Accession GSM3700103 SRX5619886 GSM3700104 SRP190015 SRS4563567 GSM3700104 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700104 GSM3700104 GEO Accession GSM3700104 SRX5619887 GSM3700105 SRP190015 SRS4563568 GSM3700105 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700105 GSM3700105 GEO Accession GSM3700105 SRX5619888 GSM3700106 SRP190015 SRS4563569 GSM3700106 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700106 GSM3700106 GEO Accession GSM3700106 SRX5619889 GSM3700107 SRP190015 SRS4563570 GSM3700107 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700107 GSM3700107 GEO Accession GSM3700107 SRX5619890 GSM3700108 SRP190015 SRS4563571 GSM3700108 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700108 GSM3700108 GEO Accession GSM3700108 SRX5619891 GSM3700109 SRP190015 SRS4563572 GSM3700109 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700109 GSM3700109 GEO Accession GSM3700109 SRX5619892 GSM3700110 SRP190015 SRS4563573 GSM3700110 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700110 GSM3700110 GEO Accession GSM3700110 SRX5619893 GSM3700111 SRP190015 SRS4563574 GSM3700111 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700111 GSM3700111 GEO Accession GSM3700111 SRX5619894 GSM3700112 SRP190015 SRS4563575 GSM3700112 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700112 GSM3700112 GEO Accession GSM3700112 SRX5619895 GSM3700113 SRP190015 SRS4563576 GSM3700113 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700113 GSM3700113 GEO Accession GSM3700113 SRX5619896 GSM3700114 SRP190015 SRS4563577 GSM3700114 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700114 GSM3700114 GEO Accession GSM3700114 SRX5619897 GSM3700115 SRP190015 SRS4563578 GSM3700115 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700115 GSM3700115 GEO Accession GSM3700115 SRX5619898 GSM3700116 SRP190015 SRS4563580 GSM3700116 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700116 GSM3700116 GEO Accession GSM3700116 SRX5619899 GSM3700117 SRP190015 SRS4563579 GSM3700117 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700117 GSM3700117 GEO Accession GSM3700117 SRX5619900 GSM3700118 SRP190015 SRS4563581 GSM3700118 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700118 GSM3700118 GEO Accession GSM3700118 SRX5619901 GSM3700119 SRP190015 SRS4563582 GSM3700119 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700119 GSM3700119 GEO Accession GSM3700119 SRX5619902 GSM3700120 SRP190015 SRS4563583 GSM3700120 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700120 GSM3700120 GEO Accession GSM3700120 SRX5619903 GSM3700121 SRP190015 SRS4563584 GSM3700121 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700121 GSM3700121 GEO Accession GSM3700121 SRX5619904 GSM3700122 SRP190015 SRS4563585 GSM3700122 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700122 GSM3700122 GEO Accession GSM3700122 SRX5619905 GSM3700123 SRP190015 SRS4563587 GSM3700123 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700123 GSM3700123 GEO Accession GSM3700123 SRX5619906 GSM3700124 SRP190015 SRS4563586 GSM3700124 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700124 GSM3700124 GEO Accession GSM3700124 SRX5619907 GSM3700125 SRP190015 SRS4566037 GSM3700125 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700125 GSM3700125 GEO Accession GSM3700125 SRX5619908 GSM3700126 SRP190015 SRS4566036 GSM3700126 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700126 GSM3700126 GEO Accession GSM3700126 SRX5619909 GSM3700127 SRP190015 SRS4566038 GSM3700127 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700127 GSM3700127 GEO Accession GSM3700127 SRX5619910 GSM3700128 SRP190015 SRS4566039 GSM3700128 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700128 GSM3700128 GEO Accession GSM3700128 SRX5619911 GSM3700129 SRP190015 SRS4566041 GSM3700129 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700129 GSM3700129 GEO Accession GSM3700129 SRX5619912 GSM3700130 SRP190015 SRS4566040 GSM3700130 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700130 GSM3700130 GEO Accession GSM3700130 SRX5619913 GSM3700131 SRP190015 SRS4566042 GSM3700131 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700131 GSM3700131 GEO Accession GSM3700131 SRX5619914 GSM3700132 SRP190015 SRS4566043 GSM3700132 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700132 GSM3700132 GEO Accession GSM3700132 SRX5619915 GSM3700133 SRP190015 SRS4566044 GSM3700133 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700133 GSM3700133 GEO Accession GSM3700133 SRX5619916 GSM3700134 SRP190015 SRS4566045 GSM3700134 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700134 GSM3700134 GEO Accession GSM3700134 SRX5619917 GSM3700135 SRP190015 SRS4566046 GSM3700135 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700135 GSM3700135 GEO Accession GSM3700135 SRX5619918 GSM3700136 SRP190015 SRS4566047 GSM3700136 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700136 GSM3700136 GEO Accession GSM3700136 SRX5619919 GSM3700137 SRP190015 SRS4566048 GSM3700137 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700137 GSM3700137 GEO Accession GSM3700137 SRX5619920 GSM3700138 SRP190015 SRS4566050 GSM3700138 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700138 GSM3700138 GEO Accession GSM3700138 SRX5619921 GSM3700139 SRP190015 SRS4566049 GSM3700139 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700139 GSM3700139 GEO Accession GSM3700139 SRX5619922 GSM3700140 SRP190015 SRS4566051 GSM3700140 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700140 GSM3700140 GEO Accession GSM3700140 SRX5619923 GSM3700141 SRP190015 SRS4566053 GSM3700141 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700141 GSM3700141 GEO Accession GSM3700141 SRX5619924 GSM3700142 SRP190015 SRS4566052 GSM3700142 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700142 GSM3700142 GEO Accession GSM3700142 SRX5619925 GSM3700143 SRP190015 SRS4566056 GSM3700143 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700143 GSM3700143 GEO Accession GSM3700143 SRX5619926 GSM3700144 SRP190015 SRS4566054 GSM3700144 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700144 GSM3700144 GEO Accession GSM3700144 SRX5619927 GSM3700145 SRP190015 SRS4566055 GSM3700145 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700145 GSM3700145 GEO Accession GSM3700145 SRX5619928 GSM3700146 SRP190015 SRS4566058 GSM3700146 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700146 GSM3700146 GEO Accession GSM3700146 SRX5619929 GSM3700147 SRP190015 SRS4566059 GSM3700147 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700147 GSM3700147 GEO Accession GSM3700147 SRX5619930 GSM3700148 SRP190015 SRS4566060 GSM3700148 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700148 GSM3700148 GEO Accession GSM3700148 SRX5619931 GSM3700149 SRP190015 SRS4566057 GSM3700149 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700149 GSM3700149 GEO Accession GSM3700149 SRX5619932 GSM3700150 SRP190015 SRS4566062 GSM3700150 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700150 GSM3700150 GEO Accession GSM3700150 SRX5619933 GSM3700151 SRP190015 SRS4566064 GSM3700151 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700151 GSM3700151 GEO Accession GSM3700151 SRX5619934 GSM3700152 SRP190015 SRS4566063 GSM3700152 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700152 GSM3700152 GEO Accession GSM3700152 SRX5619935 GSM3700153 SRP190015 SRS4566065 GSM3700153 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700153 GSM3700153 GEO Accession GSM3700153 SRX5619936 GSM3700154 SRP190015 SRS4566066 GSM3700154 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700154 GSM3700154 GEO Accession GSM3700154 SRX5619937 GSM3700155 SRP190015 SRS4566061 GSM3700155 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700155 GSM3700155 GEO Accession GSM3700155 SRX5619938 GSM3700156 SRP190015 SRS4566067 GSM3700156 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700156 GSM3700156 GEO Accession GSM3700156 SRX5619939 GSM3700157 SRP190015 SRS4566068 GSM3700157 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700157 GSM3700157 GEO Accession GSM3700157 SRX5619940 GSM3700158 SRP190015 SRS4566070 GSM3700158 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700158 GSM3700158 GEO Accession GSM3700158 SRX5619941 GSM3700159 SRP190015 SRS4566069 GSM3700159 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700159 GSM3700159 GEO Accession GSM3700159 SRX5619942 GSM3700160 SRP190015 SRS4566071 GSM3700160 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700160 GSM3700160 GEO Accession GSM3700160 SRX5619943 GSM3700161 SRP190015 SRS4566072 GSM3700161 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700161 GSM3700161 GEO Accession GSM3700161 SRX5619944 GSM3700162 SRP190015 SRS4566073 GSM3700162 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700162 GSM3700162 GEO Accession GSM3700162 SRX5619945 GSM3700163 SRP190015 SRS4566076 GSM3700163 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700163 GSM3700163 GEO Accession GSM3700163 SRX5619946 GSM3700164 SRP190015 SRS4566074 GSM3700164 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700164 GSM3700164 GEO Accession GSM3700164 SRX5619947 GSM3700165 SRP190015 SRS4566077 GSM3700165 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700165 GSM3700165 GEO Accession GSM3700165 SRX5619948 GSM3700166 SRP190015 SRS4566075 GSM3700166 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700166 GSM3700166 GEO Accession GSM3700166 SRX5619949 GSM3700167 SRP190015 SRS4566078 GSM3700167 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700167 GSM3700167 GEO Accession GSM3700167 SRX5619950 GSM3700168 SRP190015 SRS4566079 GSM3700168 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700168 GSM3700168 GEO Accession GSM3700168 SRX5619951 GSM3700169 SRP190015 SRS4566083 GSM3700169 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700169 GSM3700169 GEO Accession GSM3700169 SRX5619952 GSM3700170 SRP190015 SRS4566080 GSM3700170 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700170 GSM3700170 GEO Accession GSM3700170 SRX5619953 GSM3700171 SRP190015 SRS4566081 GSM3700171 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700171 GSM3700171 GEO Accession GSM3700171 SRX5619954 GSM3700172 SRP190015 SRS4566084 GSM3700172 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700172 GSM3700172 GEO Accession GSM3700172 SRX5619955 GSM3700173 SRP190015 SRS4566082 GSM3700173 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700173 GSM3700173 GEO Accession GSM3700173 SRX5619956 GSM3700174 SRP190015 SRS4566085 GSM3700174 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700174 GSM3700174 GEO Accession GSM3700174 SRX5619957 GSM3700175 SRP190015 SRS4566087 GSM3700175 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700175 GSM3700175 GEO Accession GSM3700175 SRX5619958 GSM3700176 SRP190015 SRS4566086 GSM3700176 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700176 GSM3700176 GEO Accession GSM3700176 SRX5619959 GSM3700177 SRP190015 SRS4566091 GSM3700177 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700177 GSM3700177 GEO Accession GSM3700177 SRX5619960 GSM3700178 SRP190015 SRS4566088 GSM3700178 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700178 GSM3700178 GEO Accession GSM3700178 SRX5619961 GSM3700179 SRP190015 SRS4566090 GSM3700179 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700179 GSM3700179 GEO Accession GSM3700179 SRX5619962 GSM3700180 SRP190015 SRS4566092 GSM3700180 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700180 GSM3700180 GEO Accession GSM3700180 SRX5619963 GSM3700181 SRP190015 SRS4566089 GSM3700181 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700181 GSM3700181 GEO Accession GSM3700181 SRX5619964 GSM3700182 SRP190015 SRS4566093 GSM3700182 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700182 GSM3700182 GEO Accession GSM3700182 SRX5619965 GSM3700183 SRP190015 SRS4566094 GSM3700183 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700183 GSM3700183 GEO Accession GSM3700183 SRX5619966 GSM3700184 SRP190015 SRS4566095 GSM3700184 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700184 GSM3700184 GEO Accession GSM3700184 SRX5619967 GSM3700185 SRP190015 SRS4566096 GSM3700185 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700185 GSM3700185 GEO Accession GSM3700185 SRX5619968 GSM3700186 SRP190015 SRS4566097 GSM3700186 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700186 GSM3700186 GEO Accession GSM3700186 SRX5619969 GSM3700187 SRP190015 SRS4566101 GSM3700187 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700187 GSM3700187 GEO Accession GSM3700187 SRX5619970 GSM3700188 SRP190015 SRS4566099 GSM3700188 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700188 GSM3700188 GEO Accession GSM3700188 SRX5619971 GSM3700189 SRP190015 SRS4566098 GSM3700189 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700189 GSM3700189 GEO Accession GSM3700189 SRX5619972 GSM3700190 SRP190015 SRS4566105 GSM3700190 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700190 GSM3700190 GEO Accession GSM3700190 SRX5619973 GSM3700191 SRP190015 SRS4566100 GSM3700191 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700191 GSM3700191 GEO Accession GSM3700191 SRX5619974 GSM3700192 SRP190015 SRS4566102 GSM3700192 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700192 GSM3700192 GEO Accession GSM3700192 SRX5619975 GSM3700193 SRP190015 SRS4566103 GSM3700193 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700193 GSM3700193 GEO Accession GSM3700193 SRX5619976 GSM3700194 SRP190015 SRS4566104 GSM3700194 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700194 GSM3700194 GEO Accession GSM3700194 SRX5619977 GSM3700195 SRP190015 SRS4566107 GSM3700195 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700195 GSM3700195 GEO Accession GSM3700195 SRX5619978 GSM3700196 SRP190015 SRS4566106 GSM3700196 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700196 GSM3700196 GEO Accession GSM3700196 SRX5619979 GSM3700197 SRP190015 SRS4566109 GSM3700197 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700197 GSM3700197 GEO Accession GSM3700197 SRX5619980 GSM3700198 SRP190015 SRS4566108 GSM3700198 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700198 GSM3700198 GEO Accession GSM3700198 SRX5619981 GSM3700199 SRP190015 SRS4566110 GSM3700199 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700199 GSM3700199 GEO Accession GSM3700199 SRX5619982 GSM3700200 SRP190015 SRS4566113 GSM3700200 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700200 GSM3700200 GEO Accession GSM3700200 SRX5619983 GSM3700201 SRP190015 SRS4566111 GSM3700201 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700201 GSM3700201 GEO Accession GSM3700201 SRX5619984 GSM3700202 SRP190015 SRS4566117 GSM3700202 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700202 GSM3700202 GEO Accession GSM3700202 SRX5619985 GSM3700203 SRP190015 SRS4566112 GSM3700203 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700203 GSM3700203 GEO Accession GSM3700203 SRX5619986 GSM3700204 SRP190015 SRS4566115 GSM3700204 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700204 GSM3700204 GEO Accession GSM3700204 SRX5619987 GSM3700205 SRP190015 SRS4566116 GSM3700205 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700205 GSM3700205 GEO Accession GSM3700205 SRX5619988 GSM3700206 SRP190015 SRS4566114 GSM3700206 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700206 GSM3700206 GEO Accession GSM3700206 SRX5619989 GSM3700207 SRP190015 SRS4566118 GSM3700207 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700207 GSM3700207 GEO Accession GSM3700207 SRX5619990 GSM3700208 SRP190015 SRS4566121 GSM3700208 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700208 GSM3700208 GEO Accession GSM3700208 SRX5619991 GSM3700209 SRP190015 SRS4566119 GSM3700209 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700209 GSM3700209 GEO Accession GSM3700209 SRX5619992 GSM3700210 SRP190015 SRS4566120 GSM3700210 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700210 GSM3700210 GEO Accession GSM3700210 SRX5619993 GSM3700211 SRP190015 SRS4566123 GSM3700211 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700211 GSM3700211 GEO Accession GSM3700211 SRX5619994 GSM3700212 SRP190015 SRS4566122 GSM3700212 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700212 GSM3700212 GEO Accession GSM3700212 SRX5619995 GSM3700213 SRP190015 SRS4566124 GSM3700213 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700213 GSM3700213 GEO Accession GSM3700213 SRX5619996 GSM3700214 SRP190015 SRS4566125 GSM3700214 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700214 GSM3700214 GEO Accession GSM3700214 SRX5619997 GSM3700215 SRP190015 SRS4566127 GSM3700215 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700215 GSM3700215 GEO Accession GSM3700215 SRX5619998 GSM3700216 SRP190015 SRS4566126 GSM3700216 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700216 GSM3700216 GEO Accession GSM3700216 SRX5619999 GSM3700217 SRP190015 SRS4566128 GSM3700217 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700217 GSM3700217 GEO Accession GSM3700217 SRX5620000 GSM3700218 SRP190015 SRS4566130 GSM3700218 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700218 GSM3700218 GEO Accession GSM3700218 SRX5620001 GSM3700219 SRP190015 SRS4566131 GSM3700219 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700219 GSM3700219 GEO Accession GSM3700219 SRX5620002 GSM3700220 SRP190015 SRS4566129 GSM3700220 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700220 GSM3700220 GEO Accession GSM3700220 SRX5620003 GSM3700221 SRP190015 SRS4566132 GSM3700221 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700221 GSM3700221 GEO Accession GSM3700221 SRX5620004 GSM3700222 SRP190015 SRS4566133 GSM3700222 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700222 GSM3700222 GEO Accession GSM3700222 SRX5620005 GSM3700223 SRP190015 SRS4566134 GSM3700223 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700223 GSM3700223 GEO Accession GSM3700223 SRX5620006 GSM3700224 SRP190015 SRS4566138 GSM3700224 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700224 GSM3700224 GEO Accession GSM3700224 SRX5620007 GSM3700225 SRP190015 SRS4566136 GSM3700225 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700225 GSM3700225 GEO Accession GSM3700225 SRX5620008 GSM3700226 SRP190015 SRS4566135 GSM3700226 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700226 GSM3700226 GEO Accession GSM3700226 SRX5620009 GSM3700227 SRP190015 SRS4566137 GSM3700227 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700227 GSM3700227 GEO Accession GSM3700227 SRX5620010 GSM3700228 SRP190015 SRS4566139 GSM3700228 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700228 GSM3700228 GEO Accession GSM3700228 SRX5620011 GSM3700229 SRP190015 SRS4566141 GSM3700229 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700229 GSM3700229 GEO Accession GSM3700229 SRX5620012 GSM3700230 SRP190015 SRS4566142 GSM3700230 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700230 GSM3700230 GEO Accession GSM3700230 SRX5620013 GSM3700231 SRP190015 SRS4566140 GSM3700231 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700231 GSM3700231 GEO Accession GSM3700231 SRX5620014 GSM3700232 SRP190015 SRS4566143 GSM3700232 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700232 GSM3700232 GEO Accession GSM3700232 SRX5620015 GSM3700233 SRP190015 SRS4566144 GSM3700233 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700233 GSM3700233 GEO Accession GSM3700233 SRX5620016 GSM3700234 SRP190015 SRS4566146 GSM3700234 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700234 GSM3700234 GEO Accession GSM3700234 SRX5620017 GSM3700235 SRP190015 SRS4566145 GSM3700235 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700235 GSM3700235 GEO Accession GSM3700235 SRX5620018 GSM3700236 SRP190015 SRS4566147 GSM3700236 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700236 GSM3700236 GEO Accession GSM3700236 SRX5620019 GSM3700237 SRP190015 SRS4566150 GSM3700237 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700237 GSM3700237 GEO Accession GSM3700237 SRX5620020 GSM3700238 SRP190015 SRS4566149 GSM3700238 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700238 GSM3700238 GEO Accession GSM3700238 SRX5620021 GSM3700239 SRP190015 SRS4566148 GSM3700239 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700239 GSM3700239 GEO Accession GSM3700239 SRX5620022 GSM3700240 SRP190015 SRS4566151 GSM3700240 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700240 GSM3700240 GEO Accession GSM3700240 SRX5620023 GSM3700241 SRP190015 SRS4566152 GSM3700241 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700241 GSM3700241 GEO Accession GSM3700241 SRX5620024 GSM3700242 SRP190015 SRS4566155 GSM3700242 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700242 GSM3700242 GEO Accession GSM3700242 SRX5620025 GSM3700243 SRP190015 SRS4566153 GSM3700243 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700243 GSM3700243 GEO Accession GSM3700243 SRX5620026 GSM3700244 SRP190015 SRS4566154 GSM3700244 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700244 GSM3700244 GEO Accession GSM3700244 SRX5620027 GSM3700245 SRP190015 SRS4566156 GSM3700245 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700245 GSM3700245 GEO Accession GSM3700245 SRX5620028 GSM3700246 SRP190015 SRS4566157 GSM3700246 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700246 GSM3700246 GEO Accession GSM3700246 SRX5620029 GSM3700247 SRP190015 SRS4566161 GSM3700247 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700247 GSM3700247 GEO Accession GSM3700247 SRX5620030 GSM3700248 SRP190015 SRS4566160 GSM3700248 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700248 GSM3700248 GEO Accession GSM3700248 SRX5620031 GSM3700249 SRP190015 SRS4566158 GSM3700249 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700249 GSM3700249 GEO Accession GSM3700249 SRX5620032 GSM3700250 SRP190015 SRS4566159 GSM3700250 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700250 GSM3700250 GEO Accession GSM3700250 SRX5620033 GSM3700251 SRP190015 SRS4566163 GSM3700251 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700251 GSM3700251 GEO Accession GSM3700251 SRX5620034 GSM3700252 SRP190015 SRS4566164 GSM3700252 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700252 GSM3700252 GEO Accession GSM3700252 SRX5620035 GSM3700253 SRP190015 SRS4566162 GSM3700253 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700253 GSM3700253 GEO Accession GSM3700253 SRX5620036 GSM3700254 SRP190015 SRS4566165 GSM3700254 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700254 GSM3700254 GEO Accession GSM3700254 SRX5620037 GSM3700255 SRP190015 SRS4566168 GSM3700255 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700255 GSM3700255 GEO Accession GSM3700255 SRX5620038 GSM3700256 SRP190015 SRS4566166 GSM3700256 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700256 GSM3700256 GEO Accession GSM3700256 SRX5620039 GSM3700257 SRP190015 SRS4566167 GSM3700257 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700257 GSM3700257 GEO Accession GSM3700257 SRX5620040 GSM3700258 SRP190015 SRS4566169 GSM3700258 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700258 GSM3700258 GEO Accession GSM3700258 SRX5620041 GSM3700259 SRP190015 SRS4566170 GSM3700259 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700259 GSM3700259 GEO Accession GSM3700259 SRX5620042 GSM3700260 SRP190015 SRS4566172 GSM3700260 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700260 GSM3700260 GEO Accession GSM3700260 SRX5620043 GSM3700261 SRP190015 SRS4566175 GSM3700261 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700261 GSM3700261 GEO Accession GSM3700261 SRX5620044 GSM3700262 SRP190015 SRS4566173 GSM3700262 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700262 GSM3700262 GEO Accession GSM3700262 SRX5620045 GSM3700263 SRP190015 SRS4566171 GSM3700263 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700263 GSM3700263 GEO Accession GSM3700263 SRX5620046 GSM3700264 SRP190015 SRS4566174 GSM3700264 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700264 GSM3700264 GEO Accession GSM3700264 SRX5620047 GSM3700265 SRP190015 SRS4566176 GSM3700265 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700265 GSM3700265 GEO Accession GSM3700265 SRX5620048 GSM3700266 SRP190015 SRS4566177 GSM3700266 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700266 GSM3700266 GEO Accession GSM3700266 SRX5620049 GSM3700267 SRP190015 SRS4566178 GSM3700267 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700267 GSM3700267 GEO Accession GSM3700267 SRX5620050 GSM3700268 SRP190015 SRS4566181 GSM3700268 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700268 GSM3700268 GEO Accession GSM3700268 SRX5620051 GSM3700269 SRP190015 SRS4566179 GSM3700269 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700269 GSM3700269 GEO Accession GSM3700269 SRX5620052 GSM3700270 SRP190015 SRS4566182 GSM3700270 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700270 GSM3700270 GEO Accession GSM3700270 SRX5620053 GSM3700271 SRP190015 SRS4566180 GSM3700271 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700271 GSM3700271 GEO Accession GSM3700271 SRX5620054 GSM3700272 SRP190015 SRS4566183 GSM3700272 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing K562 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to the remaining 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (total 479 single cells). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700272 GSM3700272 GEO Accession GSM3700272 SRX5620055 GSM3700273 SRP190015 SRS4566184 GSM3700273 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700273 GSM3700273 GEO Accession GSM3700273 SRX5620056 GSM3700274 SRP190015 SRS4566187 GSM3700274 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700274 GSM3700274 GEO Accession GSM3700274 SRX5620057 GSM3700275 SRP190015 SRS4566185 GSM3700275 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700275 GSM3700275 GEO Accession GSM3700275 SRX5620058 GSM3700276 SRP190015 SRS4566186 GSM3700276 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700276 GSM3700276 GEO Accession GSM3700276 SRX5620059 GSM3700277 SRP190015 SRS4566191 GSM3700277 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700277 GSM3700277 GEO Accession GSM3700277 SRX5620060 GSM3700278 SRP190015 SRS4566188 GSM3700278 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700278 GSM3700278 GEO Accession GSM3700278 SRX5620061 GSM3700279 SRP190015 SRS4566190 GSM3700279 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700279 GSM3700279 GEO Accession GSM3700279 SRX5620062 GSM3700280 SRP190015 SRS4566194 GSM3700280 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700280 GSM3700280 GEO Accession GSM3700280 SRX5620063 GSM3700281 SRP190015 SRS4566189 GSM3700281 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700281 GSM3700281 GEO Accession GSM3700281 SRX5620064 GSM3700282 SRP190015 SRS4566193 GSM3700282 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700282 GSM3700282 GEO Accession GSM3700282 SRX5620065 GSM3700283 SRP190015 SRS4566192 GSM3700283 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700283 GSM3700283 GEO Accession GSM3700283 SRX5620066 GSM3700284 SRP190015 SRS4566196 GSM3700284 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700284 GSM3700284 GEO Accession GSM3700284 SRX5620067 GSM3700285 SRP190015 SRS4566198 GSM3700285 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700285 GSM3700285 GEO Accession GSM3700285 SRX5620068 GSM3700286 SRP190015 SRS4566197 GSM3700286 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700286 GSM3700286 GEO Accession GSM3700286 SRX5620069 GSM3700287 SRP190015 SRS4566200 GSM3700287 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700287 GSM3700287 GEO Accession GSM3700287 SRX5620070 GSM3700288 SRP190015 SRS4566195 GSM3700288 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700288 GSM3700288 GEO Accession GSM3700288 SRX5620071 GSM3700289 SRP190015 SRS4566199 GSM3700289 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700289 GSM3700289 GEO Accession GSM3700289 SRX5620072 GSM3700290 SRP190015 SRS4566203 GSM3700290 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700290 GSM3700290 GEO Accession GSM3700290 SRX5620073 GSM3700291 SRP190015 SRS4566201 GSM3700291 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700291 GSM3700291 GEO Accession GSM3700291 SRX5620074 GSM3700292 SRP190015 SRS4566202 GSM3700292 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700292 GSM3700292 GEO Accession GSM3700292 SRX5620075 GSM3700293 SRP190015 SRS4566205 GSM3700293 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700293 GSM3700293 GEO Accession GSM3700293 SRX5620076 GSM3700294 SRP190015 SRS4566204 GSM3700294 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700294 GSM3700294 GEO Accession GSM3700294 SRX5620077 GSM3700295 SRP190015 SRS4566206 GSM3700295 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700295 GSM3700295 GEO Accession GSM3700295 SRX5620078 GSM3700296 SRP190015 SRS4566208 GSM3700296 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700296 GSM3700296 GEO Accession GSM3700296 SRX5620079 GSM3700297 SRP190015 SRS4566210 GSM3700297 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700297 GSM3700297 GEO Accession GSM3700297 SRX5620080 GSM3700298 SRP190015 SRS4566207 GSM3700298 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700298 GSM3700298 GEO Accession GSM3700298 SRX5620081 GSM3700299 SRP190015 SRS4566209 GSM3700299 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700299 GSM3700299 GEO Accession GSM3700299 SRX5620082 GSM3700300 SRP190015 SRS4566213 GSM3700300 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700300 GSM3700300 GEO Accession GSM3700300 SRX5620083 GSM3700301 SRP190015 SRS4566212 GSM3700301 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700301 GSM3700301 GEO Accession GSM3700301 SRX5620084 GSM3700302 SRP190015 SRS4566211 GSM3700302 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700302 GSM3700302 GEO Accession GSM3700302 SRX5620085 GSM3700303 SRP190015 SRS4566217 GSM3700303 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700303 GSM3700303 GEO Accession GSM3700303 SRX5620086 GSM3700304 SRP190015 SRS4566215 GSM3700304 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700304 GSM3700304 GEO Accession GSM3700304 SRX5620087 GSM3700305 SRP190015 SRS4566214 GSM3700305 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700305 GSM3700305 GEO Accession GSM3700305 SRX5620088 GSM3700306 SRP190015 SRS4566216 GSM3700306 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700306 GSM3700306 GEO Accession GSM3700306 SRX5620089 GSM3700307 SRP190015 SRS4566218 GSM3700307 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700307 GSM3700307 GEO Accession GSM3700307 SRX5620090 GSM3700308 SRP190015 SRS4566219 GSM3700308 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700308 GSM3700308 GEO Accession GSM3700308 SRX5620091 GSM3700309 SRP190015 SRS4566220 GSM3700309 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700309 GSM3700309 GEO Accession GSM3700309 SRX5620092 GSM3700310 SRP190015 SRS4566222 GSM3700310 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700310 GSM3700310 GEO Accession GSM3700310 SRX5620093 GSM3700311 SRP190015 SRS4566221 GSM3700311 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700311 GSM3700311 GEO Accession GSM3700311 SRX5620094 GSM3700312 SRP190015 SRS4566223 GSM3700312 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700312 GSM3700312 GEO Accession GSM3700312 SRX5620095 GSM3700313 SRP190015 SRS4566224 GSM3700313 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700313 GSM3700313 GEO Accession GSM3700313 SRX5620096 GSM3700314 SRP190015 SRS4566225 GSM3700314 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700314 GSM3700314 GEO Accession GSM3700314 SRX5620097 GSM3700315 SRP190015 SRS4566226 GSM3700315 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700315 GSM3700315 GEO Accession GSM3700315 SRX5620098 GSM3700316 SRP190015 SRS4566229 GSM3700316 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700316 GSM3700316 GEO Accession GSM3700316 SRX5620099 GSM3700317 SRP190015 SRS4566227 GSM3700317 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700317 GSM3700317 GEO Accession GSM3700317 SRX5620100 GSM3700318 SRP190015 SRS4566228 GSM3700318 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700318 GSM3700318 GEO Accession GSM3700318 SRX5620101 GSM3700319 SRP190015 SRS4566231 GSM3700319 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700319 GSM3700319 GEO Accession GSM3700319 SRX5620102 GSM3700320 SRP190015 SRS4566230 GSM3700320 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700320 GSM3700320 GEO Accession GSM3700320 SRX5620103 GSM3700321 SRP190015 SRS4566232 GSM3700321 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700321 GSM3700321 GEO Accession GSM3700321 SRX5620104 GSM3700322 SRP190015 SRS4566233 GSM3700322 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700322 GSM3700322 GEO Accession GSM3700322 SRX5620105 GSM3700323 SRP190015 SRS4566234 GSM3700323 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700323 GSM3700323 GEO Accession GSM3700323 SRX5620106 GSM3700324 SRP190015 SRS4566235 GSM3700324 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700324 GSM3700324 GEO Accession GSM3700324 SRX5620107 GSM3700325 SRP190015 SRS4566237 GSM3700325 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700325 GSM3700325 GEO Accession GSM3700325 SRX5620108 GSM3700326 SRP190015 SRS4566236 GSM3700326 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700326 GSM3700326 GEO Accession GSM3700326 SRX5620109 GSM3700327 SRP190015 SRS4566239 GSM3700327 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700327 GSM3700327 GEO Accession GSM3700327 SRX5620110 GSM3700328 SRP190015 SRS4566240 GSM3700328 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700328 GSM3700328 GEO Accession GSM3700328 SRX5620111 GSM3700329 SRP190015 SRS4566238 GSM3700329 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700329 GSM3700329 GEO Accession GSM3700329 SRX5620112 GSM3700330 SRP190015 SRS4566242 GSM3700330 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700330 GSM3700330 GEO Accession GSM3700330 SRX5620113 GSM3700331 SRP190015 SRS4566241 GSM3700331 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700331 GSM3700331 GEO Accession GSM3700331 SRX5620114 GSM3700332 SRP190015 SRS4566243 GSM3700332 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700332 GSM3700332 GEO Accession GSM3700332 SRX5620115 GSM3700333 SRP190015 SRS4566244 GSM3700333 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700333 GSM3700333 GEO Accession GSM3700333 SRX5620116 GSM3700334 SRP190015 SRS4566245 GSM3700334 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700334 GSM3700334 GEO Accession GSM3700334 SRX5620117 GSM3700335 SRP190015 SRS4566246 GSM3700335 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700335 GSM3700335 GEO Accession GSM3700335 SRX5620118 GSM3700336 SRP190015 SRS4566247 GSM3700336 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700336 GSM3700336 GEO Accession GSM3700336 SRX5620119 GSM3700337 SRP190015 SRS4566249 GSM3700337 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700337 GSM3700337 GEO Accession GSM3700337 SRX5620120 GSM3700338 SRP190015 SRS4566248 GSM3700338 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700338 GSM3700338 GEO Accession GSM3700338 SRX5620121 GSM3700339 SRP190015 SRS4566250 GSM3700339 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700339 GSM3700339 GEO Accession GSM3700339 SRX5620122 GSM3700340 SRP190015 SRS4566251 GSM3700340 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700340 GSM3700340 GEO Accession GSM3700340 SRX5620123 GSM3700341 SRP190015 SRS4566252 GSM3700341 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700341 GSM3700341 GEO Accession GSM3700341 SRX5620124 GSM3700342 SRP190015 SRS4566253 GSM3700342 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700342 GSM3700342 GEO Accession GSM3700342 SRX5620125 GSM3700343 SRP190015 SRS4566254 GSM3700343 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700343 GSM3700343 GEO Accession GSM3700343 SRX5620126 GSM3700344 SRP190015 SRS4566255 GSM3700344 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700344 GSM3700344 GEO Accession GSM3700344 SRX5620127 GSM3700345 SRP190015 SRS4566256 GSM3700345 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700345 GSM3700345 GEO Accession GSM3700345 SRX5620128 GSM3700346 SRP190015 SRS4566258 GSM3700346 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700346 GSM3700346 GEO Accession GSM3700346 SRX5620129 GSM3700347 SRP190015 SRS4566257 GSM3700347 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700347 GSM3700347 GEO Accession GSM3700347 SRX5620130 GSM3700348 SRP190015 SRS4566261 GSM3700348 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700348 GSM3700348 GEO Accession GSM3700348 SRX5620131 GSM3700349 SRP190015 SRS4566259 GSM3700349 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700349 GSM3700349 GEO Accession GSM3700349 SRX5620132 GSM3700350 SRP190015 SRS4566260 GSM3700350 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700350 GSM3700350 GEO Accession GSM3700350 SRX5620133 GSM3700351 SRP190015 SRS4566262 GSM3700351 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700351 GSM3700351 GEO Accession GSM3700351 SRX5620134 GSM3700352 SRP190015 SRS4566263 GSM3700352 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700352 GSM3700352 GEO Accession GSM3700352 SRX5620135 GSM3700353 SRP190015 SRS4566265 GSM3700353 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700353 GSM3700353 GEO Accession GSM3700353 SRX5620136 GSM3700354 SRP190015 SRS4566264 GSM3700354 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700354 GSM3700354 GEO Accession GSM3700354 SRX5620137 GSM3700355 SRP190015 SRS4566269 GSM3700355 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700355 GSM3700355 GEO Accession GSM3700355 SRX5620138 GSM3700356 SRP190015 SRS4566267 GSM3700356 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700356 GSM3700356 GEO Accession GSM3700356 SRX5620139 GSM3700357 SRP190015 SRS4566266 GSM3700357 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700357 GSM3700357 GEO Accession GSM3700357 SRX5620140 GSM3700358 SRP190015 SRS4566270 GSM3700358 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700358 GSM3700358 GEO Accession GSM3700358 SRX5620141 GSM3700359 SRP190015 SRS4566268 GSM3700359 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700359 GSM3700359 GEO Accession GSM3700359 SRX5620142 GSM3700360 SRP190015 SRS4566273 GSM3700360 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700360 GSM3700360 GEO Accession GSM3700360 SRX5620143 GSM3700361 SRP190015 SRS4566271 GSM3700361 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700361 GSM3700361 GEO Accession GSM3700361 SRX5620144 GSM3700362 SRP190015 SRS4566274 GSM3700362 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700362 GSM3700362 GEO Accession GSM3700362 SRX5620145 GSM3700363 SRP190015 SRS4566272 GSM3700363 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700363 GSM3700363 GEO Accession GSM3700363 SRX5620146 GSM3700364 SRP190015 SRS4566276 GSM3700364 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700364 GSM3700364 GEO Accession GSM3700364 SRX5620147 GSM3700365 SRP190015 SRS4566277 GSM3700365 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700365 GSM3700365 GEO Accession GSM3700365 SRX5620148 GSM3700366 SRP190015 SRS4566275 GSM3700366 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700366 GSM3700366 GEO Accession GSM3700366 SRX5620149 GSM3700367 SRP190015 SRS4566278 GSM3700367 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700367 GSM3700367 GEO Accession GSM3700367 SRX5620150 GSM3700368 SRP190015 SRS4566280 GSM3700368 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700368 GSM3700368 GEO Accession GSM3700368 SRX5620151 GSM3700369 SRP190015 SRS4566283 GSM3700369 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700369 GSM3700369 GEO Accession GSM3700369 SRX5620152 GSM3700370 SRP190015 SRS4566279 GSM3700370 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700370 GSM3700370 GEO Accession GSM3700370 SRX5620153 GSM3700371 SRP190015 SRS4566282 GSM3700371 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700371 GSM3700371 GEO Accession GSM3700371 SRX5620154 GSM3700372 SRP190015 SRS4566281 GSM3700372 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700372 GSM3700372 GEO Accession GSM3700372 SRX5620155 GSM3700373 SRP190015 SRS4566285 GSM3700373 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700373 GSM3700373 GEO Accession GSM3700373 SRX5620156 GSM3700374 SRP190015 SRS4566286 GSM3700374 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700374 GSM3700374 GEO Accession GSM3700374 SRX5620157 GSM3700375 SRP190015 SRS4566287 GSM3700375 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700375 GSM3700375 GEO Accession GSM3700375 SRX5620158 GSM3700376 SRP190015 SRS4566284 GSM3700376 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700376 GSM3700376 GEO Accession GSM3700376 SRX5620159 GSM3700377 SRP190015 SRS4566289 GSM3700377 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700377 GSM3700377 GEO Accession GSM3700377 SRX5620160 GSM3700378 SRP190015 SRS4566290 GSM3700378 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700378 GSM3700378 GEO Accession GSM3700378 SRX5620161 GSM3700379 SRP190015 SRS4566293 GSM3700379 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700379 GSM3700379 GEO Accession GSM3700379 SRX5620162 GSM3700380 SRP190015 SRS4566288 GSM3700380 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700380 GSM3700380 GEO Accession GSM3700380 SRX5620163 GSM3700381 SRP190015 SRS4566294 GSM3700381 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700381 GSM3700381 GEO Accession GSM3700381 SRX5620164 GSM3700382 SRP190015 SRS4566291 GSM3700382 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700382 GSM3700382 GEO Accession GSM3700382 SRX5620165 GSM3700383 SRP190015 SRS4566297 GSM3700383 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700383 GSM3700383 GEO Accession GSM3700383 SRX5620166 GSM3700384 SRP190015 SRS4566292 GSM3700384 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700384 GSM3700384 GEO Accession GSM3700384 SRX5620167 GSM3700385 SRP190015 SRS4566296 GSM3700385 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700385 GSM3700385 GEO Accession GSM3700385 SRX5620168 GSM3700386 SRP190015 SRS4566295 GSM3700386 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700386 GSM3700386 GEO Accession GSM3700386 SRX5620169 GSM3700387 SRP190015 SRS4566298 GSM3700387 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700387 GSM3700387 GEO Accession GSM3700387 SRX5620170 GSM3700388 SRP190015 SRS4566300 GSM3700388 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700388 GSM3700388 GEO Accession GSM3700388 SRX5620171 GSM3700389 SRP190015 SRS4566301 GSM3700389 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700389 GSM3700389 GEO Accession GSM3700389 SRX5620172 GSM3700390 SRP190015 SRS4566299 GSM3700390 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700390 GSM3700390 GEO Accession GSM3700390 SRX5620173 GSM3700391 SRP190015 SRS4566302 GSM3700391 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700391 GSM3700391 GEO Accession GSM3700391 SRX5620174 GSM3700392 SRP190015 SRS4566304 GSM3700392 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700392 GSM3700392 GEO Accession GSM3700392 SRX5620175 GSM3700393 SRP190015 SRS4566306 GSM3700393 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700393 GSM3700393 GEO Accession GSM3700393 SRX5620176 GSM3700394 SRP190015 SRS4566303 GSM3700394 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700394 GSM3700394 GEO Accession GSM3700394 SRX5620177 GSM3700395 SRP190015 SRS4566305 GSM3700395 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700395 GSM3700395 GEO Accession GSM3700395 SRX5620178 GSM3700396 SRP190015 SRS4566307 GSM3700396 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700396 GSM3700396 GEO Accession GSM3700396 SRX5620179 GSM3700397 SRP190015 SRS4566309 GSM3700397 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700397 GSM3700397 GEO Accession GSM3700397 SRX5620180 GSM3700398 SRP190015 SRS4566308 GSM3700398 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700398 GSM3700398 GEO Accession GSM3700398 SRX5620181 GSM3700399 SRP190015 SRS4566310 GSM3700399 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700399 GSM3700399 GEO Accession GSM3700399 SRX5620182 GSM3700400 SRP190015 SRS4566311 GSM3700400 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700400 GSM3700400 GEO Accession GSM3700400 SRX5620183 GSM3700401 SRP190015 SRS4566313 GSM3700401 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700401 GSM3700401 GEO Accession GSM3700401 SRX5620184 GSM3700402 SRP190015 SRS4566312 GSM3700402 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700402 GSM3700402 GEO Accession GSM3700402 SRX5620185 GSM3700403 SRP190015 SRS4566315 GSM3700403 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700403 GSM3700403 GEO Accession GSM3700403 SRX5620186 GSM3700404 SRP190015 SRS4566314 GSM3700404 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700404 GSM3700404 GEO Accession GSM3700404 SRX5620187 GSM3700405 SRP190015 SRS4566316 GSM3700405 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700405 GSM3700405 GEO Accession GSM3700405 SRX5620188 GSM3700406 SRP190015 SRS4566319 GSM3700406 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700406 GSM3700406 GEO Accession GSM3700406 SRX5620189 GSM3700407 SRP190015 SRS4566317 GSM3700407 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700407 GSM3700407 GEO Accession GSM3700407 SRX5620190 GSM3700408 SRP190015 SRS4566318 GSM3700408 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700408 GSM3700408 GEO Accession GSM3700408 SRX5620191 GSM3700409 SRP190015 SRS4566320 GSM3700409 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700409 GSM3700409 GEO Accession GSM3700409 SRX5620192 GSM3700410 SRP190015 SRS4566323 GSM3700410 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700410 GSM3700410 GEO Accession GSM3700410 SRX5620193 GSM3700411 SRP190015 SRS4566321 GSM3700411 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700411 GSM3700411 GEO Accession GSM3700411 SRX5620194 GSM3700412 SRP190015 SRS4566322 GSM3700412 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700412 GSM3700412 GEO Accession GSM3700412 SRX5620195 GSM3700413 SRP190015 SRS4566326 GSM3700413 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700413 GSM3700413 GEO Accession GSM3700413 SRX5620196 GSM3700414 SRP190015 SRS4566324 GSM3700414 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700414 GSM3700414 GEO Accession GSM3700414 SRX5620197 GSM3700415 SRP190015 SRS4566325 GSM3700415 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700415 GSM3700415 GEO Accession GSM3700415 SRX5620198 GSM3700416 SRP190015 SRS4566327 GSM3700416 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700416 GSM3700416 GEO Accession GSM3700416 SRX5620199 GSM3700417 SRP190015 SRS4566328 GSM3700417 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700417 GSM3700417 GEO Accession GSM3700417 SRX5620200 GSM3700418 SRP190015 SRS4566329 GSM3700418 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700418 GSM3700418 GEO Accession GSM3700418 SRX5620201 GSM3700419 SRP190015 SRS4566330 GSM3700419 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700419 GSM3700419 GEO Accession GSM3700419 SRX5620202 GSM3700420 SRP190015 SRS4566333 GSM3700420 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700420 GSM3700420 GEO Accession GSM3700420 SRX5620203 GSM3700421 SRP190015 SRS4566331 GSM3700421 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700421 GSM3700421 GEO Accession GSM3700421 SRX5620204 GSM3700422 SRP190015 SRS4566334 GSM3700422 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700422 GSM3700422 GEO Accession GSM3700422 SRX5620205 GSM3700423 SRP190015 SRS4566332 GSM3700423 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700423 GSM3700423 GEO Accession GSM3700423 SRX5620206 GSM3700424 SRP190015 SRS4566344 GSM3700424 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700424 GSM3700424 GEO Accession GSM3700424 SRX5620207 GSM3700425 SRP190015 SRS4566337 GSM3700425 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700425 GSM3700425 GEO Accession GSM3700425 SRX5620208 GSM3700426 SRP190015 SRS4566335 GSM3700426 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700426 GSM3700426 GEO Accession GSM3700426 SRX5620209 GSM3700427 SRP190015 SRS4566338 GSM3700427 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700427 GSM3700427 GEO Accession GSM3700427 SRX5620210 GSM3700428 SRP190015 SRS4566336 GSM3700428 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700428 GSM3700428 GEO Accession GSM3700428 SRX5620211 GSM3700429 SRP190015 SRS4566340 GSM3700429 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700429 GSM3700429 GEO Accession GSM3700429 SRX5620212 GSM3700430 SRP190015 SRS4566341 GSM3700430 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700430 GSM3700430 GEO Accession GSM3700430 SRX5620213 GSM3700431 SRP190015 SRS4566339 GSM3700431 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700431 GSM3700431 GEO Accession GSM3700431 SRX5620214 GSM3700432 SRP190015 SRS4566345 GSM3700432 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700432 GSM3700432 GEO Accession GSM3700432 SRX5620215 GSM3700433 SRP190015 SRS4566343 GSM3700433 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700433 GSM3700433 GEO Accession GSM3700433 SRX5620216 GSM3700434 SRP190015 SRS4566342 GSM3700434 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700434 GSM3700434 GEO Accession GSM3700434 SRX5620217 GSM3700435 SRP190015 SRS4566347 GSM3700435 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700435 GSM3700435 GEO Accession GSM3700435 SRX5620218 GSM3700436 SRP190015 SRS4566346 GSM3700436 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700436 GSM3700436 GEO Accession GSM3700436 SRX5620219 GSM3700437 SRP190015 SRS4566348 GSM3700437 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700437 GSM3700437 GEO Accession GSM3700437 SRX5620220 GSM3700438 SRP190015 SRS4566349 GSM3700438 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700438 GSM3700438 GEO Accession GSM3700438 SRX5620221 GSM3700439 SRP190015 SRS4566352 GSM3700439 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700439 GSM3700439 GEO Accession GSM3700439 SRX5620222 GSM3700440 SRP190015 SRS4566355 GSM3700440 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700440 GSM3700440 GEO Accession GSM3700440 SRX5620223 GSM3700441 SRP190015 SRS4566350 GSM3700441 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700441 GSM3700441 GEO Accession GSM3700441 SRX5620224 GSM3700442 SRP190015 SRS4566354 GSM3700442 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700442 GSM3700442 GEO Accession GSM3700442 SRX5620225 GSM3700443 SRP190015 SRS4566351 GSM3700443 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700443 GSM3700443 GEO Accession GSM3700443 SRX5620226 GSM3700444 SRP190015 SRS4566353 GSM3700444 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700444 GSM3700444 GEO Accession GSM3700444 SRX5620227 GSM3700445 SRP190015 SRS4566356 GSM3700445 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700445 GSM3700445 GEO Accession GSM3700445 SRX5620228 GSM3700446 SRP190015 SRS4566358 GSM3700446 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700446 GSM3700446 GEO Accession GSM3700446 SRX5620229 GSM3700447 SRP190015 SRS4566357 GSM3700447 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700447 GSM3700447 GEO Accession GSM3700447 SRX5620230 GSM3700448 SRP190015 SRS4566360 GSM3700448 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700448 GSM3700448 GEO Accession GSM3700448 SRX5620231 GSM3700449 SRP190015 SRS4566362 GSM3700449 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700449 GSM3700449 GEO Accession GSM3700449 SRX5620232 GSM3700450 SRP190015 SRS4566363 GSM3700450 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700450 GSM3700450 GEO Accession GSM3700450 SRX5620233 GSM3700451 SRP190015 SRS4566359 GSM3700451 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700451 GSM3700451 GEO Accession GSM3700451 SRX5620234 GSM3700452 SRP190015 SRS4566361 GSM3700452 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700452 GSM3700452 GEO Accession GSM3700452 SRX5620235 GSM3700453 SRP190015 SRS4566364 GSM3700453 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700453 GSM3700453 GEO Accession GSM3700453 SRX5620236 GSM3700454 SRP190015 SRS4566365 GSM3700454 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700454 GSM3700454 GEO Accession GSM3700454 SRX5620237 GSM3700455 SRP190015 SRS4566366 GSM3700455 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700455 GSM3700455 GEO Accession GSM3700455 SRX5620238 GSM3700456 SRP190015 SRS4566367 GSM3700456 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700456 GSM3700456 GEO Accession GSM3700456 SRX5620239 GSM3700457 SRP190015 SRS4566372 GSM3700457 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700457 GSM3700457 GEO Accession GSM3700457 SRX5620240 GSM3700458 SRP190015 SRS4566369 GSM3700458 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700458 GSM3700458 GEO Accession GSM3700458 SRX5620241 GSM3700459 SRP190015 SRS4566370 GSM3700459 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700459 GSM3700459 GEO Accession GSM3700459 SRX5620242 GSM3700460 SRP190015 SRS4566368 GSM3700460 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700460 GSM3700460 GEO Accession GSM3700460 SRX5620243 GSM3700461 SRP190015 SRS4566373 GSM3700461 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700461 GSM3700461 GEO Accession GSM3700461 SRX5620244 GSM3700462 SRP190015 SRS4566371 GSM3700462 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700462 GSM3700462 GEO Accession GSM3700462 SRX5620245 GSM3700463 SRP190015 SRS4566374 GSM3700463 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700463 GSM3700463 GEO Accession GSM3700463 SRX5620246 GSM3700464 SRP190015 SRS4566376 GSM3700464 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700464 GSM3700464 GEO Accession GSM3700464 SRX5620247 GSM3700465 SRP190015 SRS4566377 GSM3700465 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700465 GSM3700465 GEO Accession GSM3700465 SRX5620248 GSM3700466 SRP190015 SRS4566375 GSM3700466 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700466 GSM3700466 GEO Accession GSM3700466 SRX5620249 GSM3700467 SRP190015 SRS4566378 GSM3700467 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700467 GSM3700467 GEO Accession GSM3700467 SRX5620250 GSM3700468 SRP190015 SRS4566381 GSM3700468 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700468 GSM3700468 GEO Accession GSM3700468 SRX5620251 GSM3700469 SRP190015 SRS4566379 GSM3700469 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700469 GSM3700469 GEO Accession GSM3700469 SRX5620252 GSM3700470 SRP190015 SRS4566383 GSM3700470 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700470 GSM3700470 GEO Accession GSM3700470 SRX5620253 GSM3700471 SRP190015 SRS4566380 GSM3700471 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700471 GSM3700471 GEO Accession GSM3700471 SRX5620254 GSM3700472 SRP190015 SRS4566386 GSM3700472 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700472 GSM3700472 GEO Accession GSM3700472 SRX5620255 GSM3700473 SRP190015 SRS4566382 GSM3700473 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700473 GSM3700473 GEO Accession GSM3700473 SRX5620256 GSM3700474 SRP190015 SRS4566384 GSM3700474 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700474 GSM3700474 GEO Accession GSM3700474 SRX5620257 GSM3700475 SRP190015 SRS4566385 GSM3700475 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700475 GSM3700475 GEO Accession GSM3700475 SRX5620258 GSM3700476 SRP190015 SRS4566387 GSM3700476 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700476 GSM3700476 GEO Accession GSM3700476 SRX5620259 GSM3700477 SRP190015 SRS4566389 GSM3700477 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700477 GSM3700477 GEO Accession GSM3700477 SRX5620260 GSM3700478 SRP190015 SRS4566392 GSM3700478 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700478 GSM3700478 GEO Accession GSM3700478 SRX5620261 GSM3700479 SRP190015 SRS4566388 GSM3700479 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700479 GSM3700479 GEO Accession GSM3700479 SRX5620262 GSM3700480 SRP190015 SRS4566390 GSM3700480 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700480 GSM3700480 GEO Accession GSM3700480 SRX5620263 GSM3700481 SRP190015 SRS4566393 GSM3700481 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700481 GSM3700481 GEO Accession GSM3700481 SRX5620264 GSM3700482 SRP190015 SRS4566391 GSM3700482 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700482 GSM3700482 GEO Accession GSM3700482 SRX5620265 GSM3700483 SRP190015 SRS4566398 GSM3700483 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700483 GSM3700483 GEO Accession GSM3700483 SRX5620266 GSM3700484 SRP190015 SRS4566394 GSM3700484 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700484 GSM3700484 GEO Accession GSM3700484 SRX5620267 GSM3700485 SRP190015 SRS4566395 GSM3700485 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700485 GSM3700485 GEO Accession GSM3700485 SRX5620268 GSM3700486 SRP190015 SRS4566396 GSM3700486 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700486 GSM3700486 GEO Accession GSM3700486 SRX5620269 GSM3700487 SRP190015 SRS4566397 GSM3700487 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700487 GSM3700487 GEO Accession GSM3700487 SRX5620270 GSM3700488 SRP190015 SRS4566399 GSM3700488 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700488 GSM3700488 GEO Accession GSM3700488 SRX5620271 GSM3700489 SRP190015 SRS4566401 GSM3700489 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700489 GSM3700489 GEO Accession GSM3700489 SRX5620272 GSM3700490 SRP190015 SRS4566400 GSM3700490 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700490 GSM3700490 GEO Accession GSM3700490 SRX5620273 GSM3700491 SRP190015 SRS4566402 GSM3700491 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700491 GSM3700491 GEO Accession GSM3700491 SRX5620274 GSM3700492 SRP190015 SRS4566403 GSM3700492 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700492 GSM3700492 GEO Accession GSM3700492 SRX5620275 GSM3700493 SRP190015 SRS4566408 GSM3700493 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700493 GSM3700493 GEO Accession GSM3700493 SRX5620276 GSM3700494 SRP190015 SRS4566404 GSM3700494 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700494 GSM3700494 GEO Accession GSM3700494 SRX5620277 GSM3700495 SRP190015 SRS4566406 GSM3700495 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700495 GSM3700495 GEO Accession GSM3700495 SRX5620278 GSM3700496 SRP190015 SRS4566405 GSM3700496 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700496 GSM3700496 GEO Accession GSM3700496 SRX5620279 GSM3700497 SRP190015 SRS4566407 GSM3700497 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700497 GSM3700497 GEO Accession GSM3700497 SRX5620280 GSM3700498 SRP190015 SRS4566409 GSM3700498 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700498 GSM3700498 GEO Accession GSM3700498 SRX5620281 GSM3700499 SRP190015 SRS4566412 GSM3700499 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700499 GSM3700499 GEO Accession GSM3700499 SRX5620282 GSM3700500 SRP190015 SRS4566411 GSM3700500 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700500 GSM3700500 GEO Accession GSM3700500 SRX5620283 GSM3700501 SRP190015 SRS4566410 GSM3700501 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700501 GSM3700501 GEO Accession GSM3700501 SRX5620284 GSM3700502 SRP190015 SRS4566414 GSM3700502 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700502 GSM3700502 GEO Accession GSM3700502 SRX5620285 GSM3700503 SRP190015 SRS4566413 GSM3700503 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700503 GSM3700503 GEO Accession GSM3700503 SRX5620286 GSM3700504 SRP190015 SRS4566415 GSM3700504 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700504 GSM3700504 GEO Accession GSM3700504 SRX5620287 GSM3700505 SRP190015 SRS4566416 GSM3700505 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700505 GSM3700505 GEO Accession GSM3700505 SRX5620288 GSM3700506 SRP190015 SRS4566417 GSM3700506 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700506 GSM3700506 GEO Accession GSM3700506 SRX5620289 GSM3700507 SRP190015 SRS4566420 GSM3700507 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700507 GSM3700507 GEO Accession GSM3700507 SRX5620290 GSM3700508 SRP190015 SRS4566418 GSM3700508 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700508 GSM3700508 GEO Accession GSM3700508 SRX5620291 GSM3700509 SRP190015 SRS4566422 GSM3700509 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700509 GSM3700509 GEO Accession GSM3700509 SRX5620292 GSM3700510 SRP190015 SRS4566425 GSM3700510 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700510 GSM3700510 GEO Accession GSM3700510 SRX5620293 GSM3700511 SRP190015 SRS4566421 GSM3700511 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700511 GSM3700511 GEO Accession GSM3700511 SRX5620294 GSM3700512 SRP190015 SRS4566419 GSM3700512 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700512 GSM3700512 GEO Accession GSM3700512 SRX5620295 GSM3700513 SRP190015 SRS4566424 GSM3700513 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700513 GSM3700513 GEO Accession GSM3700513 SRX5620296 GSM3700514 SRP190015 SRS4566423 GSM3700514 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700514 GSM3700514 GEO Accession GSM3700514 SRX5620297 GSM3700515 SRP190015 SRS4566426 GSM3700515 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700515 GSM3700515 GEO Accession GSM3700515 SRX5620298 GSM3700516 SRP190015 SRS4566427 GSM3700516 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700516 GSM3700516 GEO Accession GSM3700516 SRX5620299 GSM3700517 SRP190015 SRS4566428 GSM3700517 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700517 GSM3700517 GEO Accession GSM3700517 SRX5620300 GSM3700518 SRP190015 SRS4566429 GSM3700518 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700518 GSM3700518 GEO Accession GSM3700518 SRX5620301 GSM3700519 SRP190015 SRS4566431 GSM3700519 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700519 GSM3700519 GEO Accession GSM3700519 SRX5620302 GSM3700520 SRP190015 SRS4566432 GSM3700520 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700520 GSM3700520 GEO Accession GSM3700520 SRX5620303 GSM3700521 SRP190015 SRS4566434 GSM3700521 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700521 GSM3700521 GEO Accession GSM3700521 SRX5620304 GSM3700522 SRP190015 SRS4566437 GSM3700522 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700522 GSM3700522 GEO Accession GSM3700522 SRX5620305 GSM3700523 SRP190015 SRS4566430 GSM3700523 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700523 GSM3700523 GEO Accession GSM3700523 SRX5620306 GSM3700524 SRP190015 SRS4566433 GSM3700524 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700524 GSM3700524 GEO Accession GSM3700524 SRX5620307 GSM3700525 SRP190015 SRS4566436 GSM3700525 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700525 GSM3700525 GEO Accession GSM3700525 SRX5620308 GSM3700526 SRP190015 SRS4566435 GSM3700526 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700526 GSM3700526 GEO Accession GSM3700526 SRX5620309 GSM3700527 SRP190015 SRS4566459 GSM3700527 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700527 GSM3700527 GEO Accession GSM3700527 SRX5620310 GSM3700528 SRP190015 SRS4566438 GSM3700528 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700528 GSM3700528 GEO Accession GSM3700528 SRX5620311 GSM3700529 SRP190015 SRS4566458 GSM3700529 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700529 GSM3700529 GEO Accession GSM3700529 SRX5620312 GSM3700530 SRP190015 SRS4566457 GSM3700530 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700530 GSM3700530 GEO Accession GSM3700530 SRX5620313 GSM3700531 SRP190015 SRS4566460 GSM3700531 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700531 GSM3700531 GEO Accession GSM3700531 SRX5620314 GSM3700532 SRP190015 SRS4566461 GSM3700532 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700532 GSM3700532 GEO Accession GSM3700532 SRX5620315 GSM3700533 SRP190015 SRS4566463 GSM3700533 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700533 GSM3700533 GEO Accession GSM3700533 SRX5620316 GSM3700534 SRP190015 SRS4566464 GSM3700534 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700534 GSM3700534 GEO Accession GSM3700534 SRX5620317 GSM3700535 SRP190015 SRS4566462 GSM3700535 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700535 GSM3700535 GEO Accession GSM3700535 SRX5620318 GSM3700536 SRP190015 SRS4566465 GSM3700536 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700536 GSM3700536 GEO Accession GSM3700536 SRX5620319 GSM3700537 SRP190015 SRS4566468 GSM3700537 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700537 GSM3700537 GEO Accession GSM3700537 SRX5620320 GSM3700538 SRP190015 SRS4566466 GSM3700538 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700538 GSM3700538 GEO Accession GSM3700538 SRX5620321 GSM3700539 SRP190015 SRS4566467 GSM3700539 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700539 GSM3700539 GEO Accession GSM3700539 SRX5620322 GSM3700540 SRP190015 SRS4566471 GSM3700540 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700540 GSM3700540 GEO Accession GSM3700540 SRX5620323 GSM3700541 SRP190015 SRS4566470 GSM3700541 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700541 GSM3700541 GEO Accession GSM3700541 SRX5620324 GSM3700542 SRP190015 SRS4566469 GSM3700542 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700542 GSM3700542 GEO Accession GSM3700542 SRX5620325 GSM3700543 SRP190015 SRS4566474 GSM3700543 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700543 GSM3700543 GEO Accession GSM3700543 SRX5620326 GSM3700544 SRP190015 SRS4566475 GSM3700544 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700544 GSM3700544 GEO Accession GSM3700544 SRX5620327 GSM3700545 SRP190015 SRS4566473 GSM3700545 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700545 GSM3700545 GEO Accession GSM3700545 SRX5620328 GSM3700546 SRP190015 SRS4566476 GSM3700546 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700546 GSM3700546 GEO Accession GSM3700546 SRX5620329 GSM3700547 SRP190015 SRS4566472 GSM3700547 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700547 GSM3700547 GEO Accession GSM3700547 SRX5620330 GSM3700548 SRP190015 SRS4566477 GSM3700548 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700548 GSM3700548 GEO Accession GSM3700548 SRX5620331 GSM3700549 SRP190015 SRS4566478 GSM3700549 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700549 GSM3700549 GEO Accession GSM3700549 SRX5620332 GSM3700550 SRP190015 SRS4566481 GSM3700550 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700550 GSM3700550 GEO Accession GSM3700550 SRX5620333 GSM3700551 SRP190015 SRS4566488 GSM3700551 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700551 GSM3700551 GEO Accession GSM3700551 SRX5620334 GSM3700552 SRP190015 SRS4566485 GSM3700552 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700552 GSM3700552 GEO Accession GSM3700552 SRX5620335 GSM3700553 SRP190015 SRS4566487 GSM3700553 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700553 GSM3700553 GEO Accession GSM3700553 SRX5620336 GSM3700554 SRP190015 SRS4566490 GSM3700554 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700554 GSM3700554 GEO Accession GSM3700554 SRX5620337 GSM3700555 SRP190015 SRS4566495 GSM3700555 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700555 GSM3700555 GEO Accession GSM3700555 SRX5620338 GSM3700556 SRP190015 SRS4566491 GSM3700556 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700556 GSM3700556 GEO Accession GSM3700556 SRX5620339 GSM3700557 SRP190015 SRS4566492 GSM3700557 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700557 GSM3700557 GEO Accession GSM3700557 SRX5620340 GSM3700558 SRP190015 SRS4566498 GSM3700558 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700558 GSM3700558 GEO Accession GSM3700558 SRX5620341 GSM3700559 SRP190015 SRS4566493 GSM3700559 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700559 GSM3700559 GEO Accession GSM3700559 SRX5620342 GSM3700560 SRP190015 SRS4566501 GSM3700560 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700560 GSM3700560 GEO Accession GSM3700560 SRX5620343 GSM3700561 SRP190015 SRS4566502 GSM3700561 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700561 GSM3700561 GEO Accession GSM3700561 SRX5620344 GSM3700562 SRP190015 SRS4566503 GSM3700562 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700562 GSM3700562 GEO Accession GSM3700562 SRX5620345 GSM3700563 SRP190015 SRS4566506 GSM3700563 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700563 GSM3700563 GEO Accession GSM3700563 SRX5620346 GSM3700564 SRP190015 SRS4566508 GSM3700564 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700564 GSM3700564 GEO Accession GSM3700564 SRX5620347 GSM3700565 SRP190015 SRS4566499 GSM3700565 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700565 GSM3700565 GEO Accession GSM3700565 SRX5620348 GSM3700566 SRP190015 SRS4566509 GSM3700566 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700566 GSM3700566 GEO Accession GSM3700566 SRX5620349 GSM3700567 SRP190015 SRS4566507 GSM3700567 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700567 GSM3700567 GEO Accession GSM3700567 SRX5620350 GSM3700568 SRP190015 SRS4566511 GSM3700568 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700568 GSM3700568 GEO Accession GSM3700568 SRX5620351 GSM3700569 SRP190015 SRS4566510 GSM3700569 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700569 GSM3700569 GEO Accession GSM3700569 SRX5620352 GSM3700570 SRP190015 SRS4566515 GSM3700570 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700570 GSM3700570 GEO Accession GSM3700570 SRX5620353 GSM3700571 SRP190015 SRS4566514 GSM3700571 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700571 GSM3700571 GEO Accession GSM3700571 SRX5620354 GSM3700572 SRP190015 SRS4566513 GSM3700572 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700572 GSM3700572 GEO Accession GSM3700572 SRX5620355 GSM3700573 SRP190015 SRS4566512 GSM3700573 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700573 GSM3700573 GEO Accession GSM3700573 SRX5620356 GSM3700574 SRP190015 SRS4566517 GSM3700574 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700574 GSM3700574 GEO Accession GSM3700574 SRX5620357 GSM3700575 SRP190015 SRS4566516 GSM3700575 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700575 GSM3700575 GEO Accession GSM3700575 SRX5620358 GSM3700576 SRP190015 SRS4566518 GSM3700576 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700576 GSM3700576 GEO Accession GSM3700576 SRX5620359 GSM3700577 SRP190015 SRS4566523 GSM3700577 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700577 GSM3700577 GEO Accession GSM3700577 SRX5620360 GSM3700578 SRP190015 SRS4566519 GSM3700578 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700578 GSM3700578 GEO Accession GSM3700578 SRX5620361 GSM3700579 SRP190015 SRS4566521 GSM3700579 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700579 GSM3700579 GEO Accession GSM3700579 SRX5620362 GSM3700580 SRP190015 SRS4566525 GSM3700580 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700580 GSM3700580 GEO Accession GSM3700580 SRX5620363 GSM3700581 SRP190015 SRS4566520 GSM3700581 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700581 GSM3700581 GEO Accession GSM3700581 SRX5620364 GSM3700582 SRP190015 SRS4566524 GSM3700582 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700582 GSM3700582 GEO Accession GSM3700582 SRX5620365 GSM3700583 SRP190015 SRS4566526 GSM3700583 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700583 GSM3700583 GEO Accession GSM3700583 SRX5620366 GSM3700584 SRP190015 SRS4566522 GSM3700584 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700584 GSM3700584 GEO Accession GSM3700584 SRX5620367 GSM3700585 SRP190015 SRS4566531 GSM3700585 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700585 GSM3700585 GEO Accession GSM3700585 SRX5620368 GSM3700586 SRP190015 SRS4566527 GSM3700586 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700586 GSM3700586 GEO Accession GSM3700586 SRX5620369 GSM3700587 SRP190015 SRS4566529 GSM3700587 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700587 GSM3700587 GEO Accession GSM3700587 SRX5620370 GSM3700588 SRP190015 SRS4566528 GSM3700588 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700588 GSM3700588 GEO Accession GSM3700588 SRX5620371 GSM3700589 SRP190015 SRS4566532 GSM3700589 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700589 GSM3700589 GEO Accession GSM3700589 SRX5620372 GSM3700590 SRP190015 SRS4566530 GSM3700590 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700590 GSM3700590 GEO Accession GSM3700590 SRX5620373 GSM3700591 SRP190015 SRS4566533 GSM3700591 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700591 GSM3700591 GEO Accession GSM3700591 SRX5620374 GSM3700592 SRP190015 SRS4566534 GSM3700592 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700592 GSM3700592 GEO Accession GSM3700592 SRX5620375 GSM3700593 SRP190015 SRS4566535 GSM3700593 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700593 GSM3700593 GEO Accession GSM3700593 SRX5620376 GSM3700594 SRP190015 SRS4566538 GSM3700594 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700594 GSM3700594 GEO Accession GSM3700594 SRX5620377 GSM3700595 SRP190015 SRS4566536 GSM3700595 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700595 GSM3700595 GEO Accession GSM3700595 SRX5620378 GSM3700596 SRP190015 SRS4566537 GSM3700596 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700596 GSM3700596 GEO Accession GSM3700596 SRX5620379 GSM3700597 SRP190015 SRS4566539 GSM3700597 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700597 GSM3700597 GEO Accession GSM3700597 SRX5620380 GSM3700598 SRP190015 SRS4566540 GSM3700598 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700598 GSM3700598 GEO Accession GSM3700598 SRX5620381 GSM3700599 SRP190015 SRS4566542 GSM3700599 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700599 GSM3700599 GEO Accession GSM3700599 SRX5620382 GSM3700600 SRP190015 SRS4566541 GSM3700600 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700600 GSM3700600 GEO Accession GSM3700600 SRX5620383 GSM3700601 SRP190015 SRS4566543 GSM3700601 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700601 GSM3700601 GEO Accession GSM3700601 SRX5620384 GSM3700602 SRP190015 SRS4566547 GSM3700602 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700602 GSM3700602 GEO Accession GSM3700602 SRX5620385 GSM3700603 SRP190015 SRS4566544 GSM3700603 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700603 GSM3700603 GEO Accession GSM3700603 SRX5620386 GSM3700604 SRP190015 SRS4566545 GSM3700604 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700604 GSM3700604 GEO Accession GSM3700604 SRX5620387 GSM3700605 SRP190015 SRS4566546 GSM3700605 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700605 GSM3700605 GEO Accession GSM3700605 SRX5620388 GSM3700606 SRP190015 SRS4566548 GSM3700606 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700606 GSM3700606 GEO Accession GSM3700606 SRX5620389 GSM3700607 SRP190015 SRS4566549 GSM3700607 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700607 GSM3700607 GEO Accession GSM3700607 SRX5620390 GSM3700608 SRP190015 SRS4566551 GSM3700608 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700608 GSM3700608 GEO Accession GSM3700608 SRX5620391 GSM3700609 SRP190015 SRS4566554 GSM3700609 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700609 GSM3700609 GEO Accession GSM3700609 SRX5620392 GSM3700610 SRP190015 SRS4566550 GSM3700610 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700610 GSM3700610 GEO Accession GSM3700610 SRX5620393 GSM3700611 SRP190015 SRS4566552 GSM3700611 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700611 GSM3700611 GEO Accession GSM3700611 SRX5620394 GSM3700612 SRP190015 SRS4566553 GSM3700612 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700612 GSM3700612 GEO Accession GSM3700612 SRX5620395 GSM3700613 SRP190015 SRS4566555 GSM3700613 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700613 GSM3700613 GEO Accession GSM3700613 SRX5620396 GSM3700614 SRP190015 SRS4566557 GSM3700614 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700614 GSM3700614 GEO Accession GSM3700614 SRX5620397 GSM3700615 SRP190015 SRS4566559 GSM3700615 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700615 GSM3700615 GEO Accession GSM3700615 SRX5620398 GSM3700616 SRP190015 SRS4566556 GSM3700616 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700616 GSM3700616 GEO Accession GSM3700616 SRX5620399 GSM3700617 SRP190015 SRS4566558 GSM3700617 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700617 GSM3700617 GEO Accession GSM3700617 SRX5620400 GSM3700618 SRP190015 SRS4566561 GSM3700618 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700618 GSM3700618 GEO Accession GSM3700618 SRX5620401 GSM3700619 SRP190015 SRS4566560 GSM3700619 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700619 GSM3700619 GEO Accession GSM3700619 SRX5620402 GSM3700620 SRP190015 SRS4566563 GSM3700620 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700620 GSM3700620 GEO Accession GSM3700620 SRX5620403 GSM3700621 SRP190015 SRS4566562 GSM3700621 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700621 GSM3700621 GEO Accession GSM3700621 SRX5620404 GSM3700622 SRP190015 SRS4566566 GSM3700622 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700622 GSM3700622 GEO Accession GSM3700622 SRX5620405 GSM3700623 SRP190015 SRS4566565 GSM3700623 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700623 GSM3700623 GEO Accession GSM3700623 SRX5620406 GSM3700624 SRP190015 SRS4566564 GSM3700624 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700624 GSM3700624 GEO Accession GSM3700624 SRX5620407 GSM3700625 SRP190015 SRS4566568 GSM3700625 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700625 GSM3700625 GEO Accession GSM3700625 SRX5620408 GSM3700626 SRP190015 SRS4566567 GSM3700626 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700626 GSM3700626 GEO Accession GSM3700626 SRX5620409 GSM3700627 SRP190015 SRS4566570 GSM3700627 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700627 GSM3700627 GEO Accession GSM3700627 SRX5620410 GSM3700628 SRP190015 SRS4566569 GSM3700628 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700628 GSM3700628 GEO Accession GSM3700628 SRX5620411 GSM3700629 SRP190015 SRS4566573 GSM3700629 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700629 GSM3700629 GEO Accession GSM3700629 SRX5620412 GSM3700630 SRP190015 SRS4566574 GSM3700630 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700630 GSM3700630 GEO Accession GSM3700630 SRX5620413 GSM3700631 SRP190015 SRS4566571 GSM3700631 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700631 GSM3700631 GEO Accession GSM3700631 SRX5620414 GSM3700632 SRP190015 SRS4566575 GSM3700632 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700632 GSM3700632 GEO Accession GSM3700632 SRX5620415 GSM3700633 SRP190015 SRS4566572 GSM3700633 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700633 GSM3700633 GEO Accession GSM3700633 SRX5620416 GSM3700634 SRP190015 SRS4566576 GSM3700634 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700634 GSM3700634 GEO Accession GSM3700634 SRX5620417 GSM3700635 SRP190015 SRS4566578 GSM3700635 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700635 GSM3700635 GEO Accession GSM3700635 SRX5620418 GSM3700636 SRP190015 SRS4566577 GSM3700636 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700636 GSM3700636 GEO Accession GSM3700636 SRX5620419 GSM3700637 SRP190015 SRS4566580 GSM3700637 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700637 GSM3700637 GEO Accession GSM3700637 SRX5620420 GSM3700638 SRP190015 SRS4566579 GSM3700638 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700638 GSM3700638 GEO Accession GSM3700638 SRX5620421 GSM3700639 SRP190015 SRS4566581 GSM3700639 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700639 GSM3700639 GEO Accession GSM3700639 SRX5620422 GSM3700640 SRP190015 SRS4566583 GSM3700640 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700640 GSM3700640 GEO Accession GSM3700640 SRX5620423 GSM3700641 SRP190015 SRS4566584 GSM3700641 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700641 GSM3700641 GEO Accession GSM3700641 SRX5620424 GSM3700642 SRP190015 SRS4566582 GSM3700642 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700642 GSM3700642 GEO Accession GSM3700642 SRX5620425 GSM3700643 SRP190015 SRS4566585 GSM3700643 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700643 GSM3700643 GEO Accession GSM3700643 SRX5620426 GSM3700644 SRP190015 SRS4566589 GSM3700644 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700644 GSM3700644 GEO Accession GSM3700644 SRX5620427 GSM3700645 SRP190015 SRS4566586 GSM3700645 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700645 GSM3700645 GEO Accession GSM3700645 SRX5620428 GSM3700646 SRP190015 SRS4566587 GSM3700646 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700646 GSM3700646 GEO Accession GSM3700646 SRX5620429 GSM3700647 SRP190015 SRS4566590 GSM3700647 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700647 GSM3700647 GEO Accession GSM3700647 SRX5620430 GSM3700648 SRP190015 SRS4566588 GSM3700648 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700648 GSM3700648 GEO Accession GSM3700648 SRX5620431 GSM3700649 SRP190015 SRS4566591 GSM3700649 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700649 GSM3700649 GEO Accession GSM3700649 SRX5620432 GSM3700650 SRP190015 SRS4566592 GSM3700650 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700650 GSM3700650 GEO Accession GSM3700650 SRX5620433 GSM3700651 SRP190015 SRS4566593 GSM3700651 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700651 GSM3700651 GEO Accession GSM3700651 SRX5620434 GSM3700652 SRP190015 SRS4566594 GSM3700652 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700652 GSM3700652 GEO Accession GSM3700652 SRX5620435 GSM3700653 SRP190015 SRS4566597 GSM3700653 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700653 GSM3700653 GEO Accession GSM3700653 SRX5620436 GSM3700654 SRP190015 SRS4566595 GSM3700654 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700654 GSM3700654 GEO Accession GSM3700654 SRX5620437 GSM3700655 SRP190015 SRS4566596 GSM3700655 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700655 GSM3700655 GEO Accession GSM3700655 SRX5620438 GSM3700656 SRP190015 SRS4566599 GSM3700656 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700656 GSM3700656 GEO Accession GSM3700656 SRX5620439 GSM3700657 SRP190015 SRS4566600 GSM3700657 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700657 GSM3700657 GEO Accession GSM3700657 SRX5620440 GSM3700658 SRP190015 SRS4566598 GSM3700658 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700658 GSM3700658 GEO Accession GSM3700658 SRX5620441 GSM3700659 SRP190015 SRS4566602 GSM3700659 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700659 GSM3700659 GEO Accession GSM3700659 SRX5620442 GSM3700660 SRP190015 SRS4566601 GSM3700660 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700660 GSM3700660 GEO Accession GSM3700660 SRX5620443 GSM3700661 SRP190015 SRS4566603 GSM3700661 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700661 GSM3700661 GEO Accession GSM3700661 SRX5620444 GSM3700662 SRP190015 SRS4566605 GSM3700662 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700662 GSM3700662 GEO Accession GSM3700662 SRX5620445 GSM3700663 SRP190015 SRS4566616 GSM3700663 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700663 GSM3700663 GEO Accession GSM3700663 SRX5620446 GSM3700664 SRP190015 SRS4566606 GSM3700664 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700664 GSM3700664 GEO Accession GSM3700664 SRX5620447 GSM3700665 SRP190015 SRS4566604 GSM3700665 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700665 GSM3700665 GEO Accession GSM3700665 SRX5620448 GSM3700666 SRP190015 SRS4566609 GSM3700666 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700666 GSM3700666 GEO Accession GSM3700666 SRX5620449 GSM3700667 SRP190015 SRS4566610 GSM3700667 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700667 GSM3700667 GEO Accession GSM3700667 SRX5620450 GSM3700668 SRP190015 SRS4566612 GSM3700668 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700668 GSM3700668 GEO Accession GSM3700668 SRX5620451 GSM3700669 SRP190015 SRS4566611 GSM3700669 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700669 GSM3700669 GEO Accession GSM3700669 SRX5620452 GSM3700670 SRP190015 SRS4566607 GSM3700670 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700670 GSM3700670 GEO Accession GSM3700670 SRX5620453 GSM3700671 SRP190015 SRS4566608 GSM3700671 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700671 GSM3700671 GEO Accession GSM3700671 SRX5620454 GSM3700672 SRP190015 SRS4566613 GSM3700672 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700672 GSM3700672 GEO Accession GSM3700672 SRX5620455 GSM3700673 SRP190015 SRS4566614 GSM3700673 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700673 GSM3700673 GEO Accession GSM3700673 SRX5620456 GSM3700674 SRP190015 SRS4566615 GSM3700674 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700674 GSM3700674 GEO Accession GSM3700674 SRX5620457 GSM3700675 SRP190015 SRS4566619 GSM3700675 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700675 GSM3700675 GEO Accession GSM3700675 SRX5620458 GSM3700676 SRP190015 SRS4566617 GSM3700676 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700676 GSM3700676 GEO Accession GSM3700676 SRX5620459 GSM3700677 SRP190015 SRS4566623 GSM3700677 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700677 GSM3700677 GEO Accession GSM3700677 SRX5620460 GSM3700678 SRP190015 SRS4566620 GSM3700678 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700678 GSM3700678 GEO Accession GSM3700678 SRX5620461 GSM3700679 SRP190015 SRS4566618 GSM3700679 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700679 GSM3700679 GEO Accession GSM3700679 SRX5620462 GSM3700680 SRP190015 SRS4566621 GSM3700680 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700680 GSM3700680 GEO Accession GSM3700680 SRX5620463 GSM3700681 SRP190015 SRS4566622 GSM3700681 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700681 GSM3700681 GEO Accession GSM3700681 SRX5620464 GSM3700682 SRP190015 SRS4566625 GSM3700682 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700682 GSM3700682 GEO Accession GSM3700682 SRX5620465 GSM3700683 SRP190015 SRS4566624 GSM3700683 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700683 GSM3700683 GEO Accession GSM3700683 SRX5620466 GSM3700684 SRP190015 SRS4566627 GSM3700684 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700684 GSM3700684 GEO Accession GSM3700684 SRX5620467 GSM3700685 SRP190015 SRS4566630 GSM3700685 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700685 GSM3700685 GEO Accession GSM3700685 SRX5620468 GSM3700686 SRP190015 SRS4566626 GSM3700686 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700686 GSM3700686 GEO Accession GSM3700686 SRX5620469 GSM3700687 SRP190015 SRS4566631 GSM3700687 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700687 GSM3700687 GEO Accession GSM3700687 SRX5620470 GSM3700688 SRP190015 SRS4566629 GSM3700688 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700688 GSM3700688 GEO Accession GSM3700688 SRX5620471 GSM3700689 SRP190015 SRS4566628 GSM3700689 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700689 GSM3700689 GEO Accession GSM3700689 SRX5620472 GSM3700690 SRP190015 SRS4566632 GSM3700690 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700690 GSM3700690 GEO Accession GSM3700690 SRX5620473 GSM3700691 SRP190015 SRS4566633 GSM3700691 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700691 GSM3700691 GEO Accession GSM3700691 SRX5620474 GSM3700692 SRP190015 SRS4566635 GSM3700692 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700692 GSM3700692 GEO Accession GSM3700692 SRX5620475 GSM3700693 SRP190015 SRS4566634 GSM3700693 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700693 GSM3700693 GEO Accession GSM3700693 SRX5620476 GSM3700694 SRP190015 SRS4566637 GSM3700694 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700694 GSM3700694 GEO Accession GSM3700694 SRX5620477 GSM3700695 SRP190015 SRS4566636 GSM3700695 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700695 GSM3700695 GEO Accession GSM3700695 SRX5620478 GSM3700696 SRP190015 SRS4566638 GSM3700696 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700696 GSM3700696 GEO Accession GSM3700696 SRX5620479 GSM3700697 SRP190015 SRS4566639 GSM3700697 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700697 GSM3700697 GEO Accession GSM3700697 SRX5620480 GSM3700698 SRP190015 SRS4566640 GSM3700698 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700698 GSM3700698 GEO Accession GSM3700698 SRX5620481 GSM3700699 SRP190015 SRS4566643 GSM3700699 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700699 GSM3700699 GEO Accession GSM3700699 SRX5620482 GSM3700700 SRP190015 SRS4566641 GSM3700700 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700700 GSM3700700 GEO Accession GSM3700700 SRX5620483 GSM3700701 SRP190015 SRS4566645 GSM3700701 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700701 GSM3700701 GEO Accession GSM3700701 SRX5620484 GSM3700702 SRP190015 SRS4566642 GSM3700702 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700702 GSM3700702 GEO Accession GSM3700702 SRX5620485 GSM3700703 SRP190015 SRS4566646 GSM3700703 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700703 GSM3700703 GEO Accession GSM3700703 SRX5620486 GSM3700704 SRP190015 SRS4566647 GSM3700704 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700704 GSM3700704 GEO Accession GSM3700704 SRX5620487 GSM3700705 SRP190015 SRS4566644 GSM3700705 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700705 GSM3700705 GEO Accession GSM3700705 SRX5620488 GSM3700706 SRP190015 SRS4566653 GSM3700706 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700706 GSM3700706 GEO Accession GSM3700706 SRX5620489 GSM3700707 SRP190015 SRS4566649 GSM3700707 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700707 GSM3700707 GEO Accession GSM3700707 SRX5620490 GSM3700708 SRP190015 SRS4566648 GSM3700708 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700708 GSM3700708 GEO Accession GSM3700708 SRX5620491 GSM3700709 SRP190015 SRS4566651 GSM3700709 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700709 GSM3700709 GEO Accession GSM3700709 SRX5620492 GSM3700710 SRP190015 SRS4566652 GSM3700710 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700710 GSM3700710 GEO Accession GSM3700710 SRX5620493 GSM3700711 SRP190015 SRS4566650 GSM3700711 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700711 GSM3700711 GEO Accession GSM3700711 SRX5620494 GSM3700712 SRP190015 SRS4566656 GSM3700712 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700712 GSM3700712 GEO Accession GSM3700712 SRX5620495 GSM3700713 SRP190015 SRS4566658 GSM3700713 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700713 GSM3700713 GEO Accession GSM3700713 SRX5620496 GSM3700714 SRP190015 SRS4566654 GSM3700714 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700714 GSM3700714 GEO Accession GSM3700714 SRX5620497 GSM3700715 SRP190015 SRS4566655 GSM3700715 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700715 GSM3700715 GEO Accession GSM3700715 SRX5620498 GSM3700716 SRP190015 SRS4566657 GSM3700716 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700716 GSM3700716 GEO Accession GSM3700716 SRX5620499 GSM3700717 SRP190015 SRS4566660 GSM3700717 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700717 GSM3700717 GEO Accession GSM3700717 SRX5620500 GSM3700718 SRP190015 SRS4566661 GSM3700718 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700718 GSM3700718 GEO Accession GSM3700718 SRX5620501 GSM3700719 SRP190015 SRS4566659 GSM3700719 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700719 GSM3700719 GEO Accession GSM3700719 SRX5620502 GSM3700720 SRP190015 SRS4566664 GSM3700720 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700720 GSM3700720 GEO Accession GSM3700720 SRX5620503 GSM3700721 SRP190015 SRS4566662 GSM3700721 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700721 GSM3700721 GEO Accession GSM3700721 SRX5620504 GSM3700722 SRP190015 SRS4566666 GSM3700722 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700722 GSM3700722 GEO Accession GSM3700722 SRX5620505 GSM3700723 SRP190015 SRS4566667 GSM3700723 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700723 GSM3700723 GEO Accession GSM3700723 SRX5620506 GSM3700724 SRP190015 SRS4566663 GSM3700724 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700724 GSM3700724 GEO Accession GSM3700724 SRX5620507 GSM3700725 SRP190015 SRS4566665 GSM3700725 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700725 GSM3700725 GEO Accession GSM3700725 SRX5620508 GSM3700726 SRP190015 SRS4566671 GSM3700726 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700726 GSM3700726 GEO Accession GSM3700726 SRX5620509 GSM3700727 SRP190015 SRS4566668 GSM3700727 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700727 GSM3700727 GEO Accession GSM3700727 SRX5620510 GSM3700728 SRP190015 SRS4566669 GSM3700728 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700728 GSM3700728 GEO Accession GSM3700728 SRX5620511 GSM3700729 SRP190015 SRS4566670 GSM3700729 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700729 GSM3700729 GEO Accession GSM3700729 SRX5620512 GSM3700730 SRP190015 SRS4566672 GSM3700730 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700730 GSM3700730 GEO Accession GSM3700730 SRX5620513 GSM3700731 SRP190015 SRS4566674 GSM3700731 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700731 GSM3700731 GEO Accession GSM3700731 SRX5620514 GSM3700732 SRP190015 SRS4566675 GSM3700732 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700732 GSM3700732 GEO Accession GSM3700732 SRX5620515 GSM3700733 SRP190015 SRS4566677 GSM3700733 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700733 GSM3700733 GEO Accession GSM3700733 SRX5620516 GSM3700734 SRP190015 SRS4566673 GSM3700734 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700734 GSM3700734 GEO Accession GSM3700734 SRX5620517 GSM3700735 SRP190015 SRS4566676 GSM3700735 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700735 GSM3700735 GEO Accession GSM3700735 SRX5620518 GSM3700736 SRP190015 SRS4566679 GSM3700736 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700736 GSM3700736 GEO Accession GSM3700736 SRX5620519 GSM3700737 SRP190015 SRS4566680 GSM3700737 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700737 GSM3700737 GEO Accession GSM3700737 SRX5620520 GSM3700738 SRP190015 SRS4566681 GSM3700738 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700738 GSM3700738 GEO Accession GSM3700738 SRX5620521 GSM3700739 SRP190015 SRS4566004 GSM3700739 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700739 GSM3700739 GEO Accession GSM3700739 SRX5620522 GSM3700740 SRP190015 SRS4566678 GSM3700740 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700740 GSM3700740 GEO Accession GSM3700740 SRX5620523 GSM3700741 SRP190015 SRS4566683 GSM3700741 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700741 GSM3700741 GEO Accession GSM3700741 SRX5620524 GSM3700742 SRP190015 SRS4566684 GSM3700742 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700742 GSM3700742 GEO Accession GSM3700742 SRX5620525 GSM3700743 SRP190015 SRS4566682 GSM3700743 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700743 GSM3700743 GEO Accession GSM3700743 SRX5620526 GSM3700744 SRP190015 SRS4566686 GSM3700744 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700744 GSM3700744 GEO Accession GSM3700744 SRX5620527 GSM3700745 SRP190015 SRS4566685 GSM3700745 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700745 GSM3700745 GEO Accession GSM3700745 SRX5620528 GSM3700746 SRP190015 SRS4566687 GSM3700746 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700746 GSM3700746 GEO Accession GSM3700746 SRX5620529 GSM3700747 SRP190015 SRS4566688 GSM3700747 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700747 GSM3700747 GEO Accession GSM3700747 SRX5620530 GSM3700748 SRP190015 SRS4566693 GSM3700748 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700748 GSM3700748 GEO Accession GSM3700748 SRX5620531 GSM3700749 SRP190015 SRS4566690 GSM3700749 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700749 GSM3700749 GEO Accession GSM3700749 SRX5620532 GSM3700750 SRP190015 SRS4566689 GSM3700750 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700750 GSM3700750 GEO Accession GSM3700750 SRX5620533 GSM3700751 SRP190015 SRS4566692 GSM3700751 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700751 GSM3700751 GEO Accession GSM3700751 SRX5620534 GSM3700752 SRP190015 SRS4566691 GSM3700752 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700752 GSM3700752 GEO Accession GSM3700752 SRX5620535 GSM3700753 SRP190015 SRS4566695 GSM3700753 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700753 GSM3700753 GEO Accession GSM3700753 SRX5620536 GSM3700754 SRP190015 SRS4566696 GSM3700754 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700754 GSM3700754 GEO Accession GSM3700754 SRX5620537 GSM3700755 SRP190015 SRS4566694 GSM3700755 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700755 GSM3700755 GEO Accession GSM3700755 SRX5620538 GSM3700756 SRP190015 SRS4566698 GSM3700756 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700756 GSM3700756 GEO Accession GSM3700756 SRX5620539 GSM3700757 SRP190015 SRS4566697 GSM3700757 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700757 GSM3700757 GEO Accession GSM3700757 SRX5620540 GSM3700758 SRP190015 SRS4566699 GSM3700758 OTHER GENOMIC other Single-cell CUT&Tag: Approximately 1 million exponentially growing H1 cells were processed by centrifugation between buffer and reagent exchanges in low-retention tubes throughout. Centrifugations were performed at 600g for 3 min in a swinging bucket rotor for the initial wash and incubation steps, and then at 300g for 3 min after pA-Tn5 binding. Cells were collected and washed twice with 1ml Wash Buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at room temperature. Nuclei were isolated by permeabilizing cells in NP40-Digitonin Wash Buffer (0.01% NP40, 0.01% Digitonin in wash buffer) and resuspended in 0.25 mL of NP40-Digitonin Wash buffer with 1mM EDTA. Antibody was added at a 1:50 dilution and incubated on a rotator at 4°C overnight. Permeabilized cells were then rinsed once with NP40-Digitonin Wash buffer and incubated with anti-Rabbit IgG antibody (1:50 dilution) in 0.5 ml of NP40-Digitonin Wash buffer on a rotator at room temperature for 30 min. Nuclei were then washed 3x for 5 min in 1 ml NP40-Digitonin Wash buffer to remove unbound antibodies. For pA-Tn5 binding, a 1:200 dilution of pA-Tn5 adapter complex was prepared in 0.3 mL NP40-Dig-med-buffer (0.01% NP40, 0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail), and permeabilized cells were incubated with the pA-Tn5 adapter complex on a rotator at RT for 1 hr. Cells were washed 3x for 5 min in 1 ml NP40-Dig-med-buffer to remove excess pA-Tn5 protein. Cells were resuspended in 150 µL Tagmentation buffer (10 mM MgCl2 in NP40-Dig-med-buffer) and incubated at 37 for 1h. Tagmentation was stopped by adding 50 µL of 4X Stop Buffer (40.4 mM EDTA and 2 mg/ml DAPI) and the sample was held on ice for 30 minutes. The SMARTer ICELL8 single-cell system (Takara Bio USA, Cat. #640000) was used to array single cells as described for scATAC-seq12. DAPI-stained nuclei were visualized under the microscope and if there were clumps, they were strained through 10 micron cell strainers. Cells were counted using a hematocytometer and diluted at 28 cells/µL in 0.5X PBS and 1X Second Diluent (Takara Bio USA, Cat. # 640196). 10000 cells were loaded to 4 wells of the source loading plate. Control wells containing 0.5X PBS (25 µl) and fiducial mix (25 µl) (Takara Bio USA, Cat. #640196) were also included in the source loading plate. Using the ICELL8 MultiSample NanoDispenser (MSND) FLA program, cells were dispensed into a SMARTer ICELL8 250v chip (Takara Bio USA, Cat. # 640183) at 35 nanoliter per well. After cell dispense was complete, chips were sealed with the imaging film (Takara Bio USA, Cat. #640109) and centrifuged at 400 xg for 5 min at room temperature and imaged using the ICELL8 imaging station (Takara Bio USA). Images were analyzed using automated microscopy image analysis software (CellSelect, Takara Bio USA). Since cells were stained only with DAPI, they were propidium iodide negative, so that permeabilized cells would not be excluded by default software settings. Additional single cells were manually selected for dispensing using a manual triaging procedure (~486 single cells in total). Immediately following imaging, the filter file, which notes single-cell containing wells and control wells, was generated. All of the following reagents were added to the selected set of wells which contained single cells. To index the whole chip, 72 i5 and 72 i7 unique indices [Buenostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-U264 (2015)] were dispensed at 7.32 µM using ICELL8 MSND FLA program using the index 1 and index 2 filtered dispense tool respectively at 35 nanoliter per well. KAPA PCR mix (168 ul 5X HiFi Buffer + 25.2 ul 10mM dNTPs + 8.4 ul KAPA HiFi Polymerase non-hot start + 8.4 ul NF-water) was dispensed to selected wells using ICELL8 MSND FLA program at 35 nanoliter per well. The chip was sealed and centrifuged at 2250 xg at 4◦C for at least 5 min after each dispense. The chip was sealed with a TE Sealing film (Takara Bio USA, Cat. #640109) and on-chip PCR was performed using a SMARTer ICELL8 Thermal Cycler (Takara Bio USA) as follows: 5 min at 72 ◦C and 1 min at 98 ◦C followed by 15 cycles of 30 sec at 98 ◦C, 5 sec at 55 ◦C, with a final extension at 72 ◦C for 1 min. PCR products were collected by centrifugation at ~2250 xg for 20 min using the supplied SMARTer ICELL8 Collection Kit (Takara Bio USA, Cat.#640048). Pooled libraries were purified using Ampure XP beads (Beckman Counter) in a 1:1.1 ratio. Briefly, libraries were incubated with beads for 15 min at RT, washed twice in 80% ethanol, and eluted in 10 mM Tris pH 8.0. Paired-end 25x8x8x25 bp Illumina sequencing was performed on the pooled barcoded libraries following the manufacturer's instructions. Single-cell CUT&Tag Illumina HiSeq 2500 gds 303700758 GSM3700758 GEO Accession GSM3700758