SRX15052672 GSM6080943_r1 SRP372884 PRJNA833451 SRS12798362 GSM6080943 GSM6080943 OTHER GENOMIC other 10 million SEM Cells were resuspended in 10mL fresh cell media, and crosslinked by 2% formaldhyde for 10 min with rotating at room temperature. Crosslinking were quenched by glycine to a final concentration of 0.2M and incubating for 5 min with rotating at room temperature. Cells were washed once by 1 mL of cold PBS, flash-froze cell pellets in dry ice/ethanol bath and store at -80°C HIChIP was performed as previously described in (Mumbach et. al. 2017) with the following modifications. 15 μl of 25U/μl MboI restriction enzyme (New England Biolabs, R0147M) was used for digesting chromatin from 10 million cells at 37°C for 2 hours with shaking. Proximity ligation was performed overnight at 16°C on Eppendorf thermomixer with 900rpm shaking speed. Post proximity ligated products were washed once by 1 mL of Covaris shearing buffer (10mM Tris-HCl pH8.0, 1mM EDTA, 0.1% SDS, 1x cOmplete EDTA-free protease inhibitor) and resuspended in 1mL Covaris shearing buffer then transfer to Covaris milliTUBE. Sonication was performed with Covaris LE220-plus with the following settings: Duty Cycle = 30%, PIP = 400, Cycles/Burst = 200, Dithering = x +- 0.5mm, Y +- 0.5mm, z +- 0.0mm, speed 10mm/s, pause duration 0.0 and time = 10min. 120 μl DiaMag protein G beads (diagenode, C03010021) were pre-blocked in ChIP dilution buffer plus 0.1% BSA overnight at 4°C. After Covaris shearing, add 3 mL ChIP dilution buffer plus protease inhibitor to 1 mL of sheared chromatin, then precleared with 60ul pre-blocked DiaMag protein G beads at 4°C for 1 hour. 8 μg of Diagenode CTCF antibody (C15410210-50, 2.3 μg/μl) was used for 10 million cells and rotated at 4 °C overnight. Another pre-blocked 60 μl DiaMag protein G beads were added next day and incubate at 4 °C for 3 hours. Post ChIP DNA was purified by Qiagen MinElute PCR Purification Kit, typically yield 2-11ng. HiChIP libraries were prepared using Kapa HyperPrep Kit (Roche, KK8502). 15 μl of Dynabeads MyOne Streptavidin T1 (Thermo Fisher, 65602) were used for biotin pull down. After end repair and A-tailing, 5 μl of Illumina TruSeq DNA UD Indexes were used for adaptor ligation at 20°C for 15min. Libraries were amplified for 12 to 13 PCR cycles (DNA>5ng: 12 cycles, DNA<5ng: 13 cycles). NextSeq 500 SRX15052673 GSM6080942_r1 SRP372884 PRJNA833451 SRS12798363 GSM6080942 GSM6080942 OTHER GENOMIC other 10 million SEM Cells were resuspended in 10mL fresh cell media, and crosslinked by 2% formaldhyde for 10 min with rotating at room temperature. Crosslinking were quenched by glycine to a final concentration of 0.2M and incubating for 5 min with rotating at room temperature. Cells were washed once by 1 mL of cold PBS, flash-froze cell pellets in dry ice/ethanol bath and store at -80°C HIChIP was performed as previously described in (Mumbach et. al. 2017) with the following modifications. 15 μl of 25U/μl MboI restriction enzyme (New England Biolabs, R0147M) was used for digesting chromatin from 10 million cells at 37°C for 2 hours with shaking. Proximity ligation was performed overnight at 16°C on Eppendorf thermomixer with 900rpm shaking speed. Post proximity ligated products were washed once by 1 mL of Covaris shearing buffer (10mM Tris-HCl pH8.0, 1mM EDTA, 0.1% SDS, 1x cOmplete EDTA-free protease inhibitor) and resuspended in 1mL Covaris shearing buffer then transfer to Covaris milliTUBE. Sonication was performed with Covaris LE220-plus with the following settings: Duty Cycle = 30%, PIP = 400, Cycles/Burst = 200, Dithering = x +- 0.5mm, Y +- 0.5mm, z +- 0.0mm, speed 10mm/s, pause duration 0.0 and time = 10min. 120 μl DiaMag protein G beads (diagenode, C03010021) were pre-blocked in ChIP dilution buffer plus 0.1% BSA overnight at 4°C. After Covaris shearing, add 3 mL ChIP dilution buffer plus protease inhibitor to 1 mL of sheared chromatin, then precleared with 60ul pre-blocked DiaMag protein G beads at 4°C for 1 hour. 8 μg of Diagenode CTCF antibody (C15410210-50, 2.3 μg/μl) was used for 10 million cells and rotated at 4 °C overnight. Another pre-blocked 60 μl DiaMag protein G beads were added next day and incubate at 4 °C for 3 hours. Post ChIP DNA was purified by Qiagen MinElute PCR Purification Kit, typically yield 2-11ng. HiChIP libraries were prepared using Kapa HyperPrep Kit (Roche, KK8502). 15 μl of Dynabeads MyOne Streptavidin T1 (Thermo Fisher, 65602) were used for biotin pull down. After end repair and A-tailing, 5 μl of Illumina TruSeq DNA UD Indexes were used for adaptor ligation at 20°C for 15min. Libraries were amplified for 12 to 13 PCR cycles (DNA>5ng: 12 cycles, DNA<5ng: 13 cycles). NextSeq 500 SRX15052674 GSM6080944_r1 SRP372884 PRJNA833451 SRS12798364 GSM6080944 GSM6080944 OTHER GENOMIC other 10 million SEM Cells were resuspended in 10mL fresh cell media, and crosslinked by 2% formaldhyde for 10 min with rotating at room temperature. Crosslinking were quenched by glycine to a final concentration of 0.2M and incubating for 5 min with rotating at room temperature. Cells were washed once by 1 mL of cold PBS, flash-froze cell pellets in dry ice/ethanol bath and store at -80°C HIChIP was performed as previously described in (Mumbach et. al. 2017) with the following modifications. 15 μl of 25U/μl MboI restriction enzyme (New England Biolabs, R0147M) was used for digesting chromatin from 10 million cells at 37°C for 2 hours with shaking. Proximity ligation was performed overnight at 16°C on Eppendorf thermomixer with 900rpm shaking speed. Post proximity ligated products were washed once by 1 mL of Covaris shearing buffer (10mM Tris-HCl pH8.0, 1mM EDTA, 0.1% SDS, 1x cOmplete EDTA-free protease inhibitor) and resuspended in 1mL Covaris shearing buffer then transfer to Covaris milliTUBE. Sonication was performed with Covaris LE220-plus with the following settings: Duty Cycle = 30%, PIP = 400, Cycles/Burst = 200, Dithering = x +- 0.5mm, Y +- 0.5mm, z +- 0.0mm, speed 10mm/s, pause duration 0.0 and time = 10min. 120 μl DiaMag protein G beads (diagenode, C03010021) were pre-blocked in ChIP dilution buffer plus 0.1% BSA overnight at 4°C. After Covaris shearing, add 3 mL ChIP dilution buffer plus protease inhibitor to 1 mL of sheared chromatin, then precleared with 60ul pre-blocked DiaMag protein G beads at 4°C for 1 hour. 8 μg of Diagenode CTCF antibody (C15410210-50, 2.3 μg/μl) was used for 10 million cells and rotated at 4 °C overnight. Another pre-blocked 60 μl DiaMag protein G beads were added next day and incubate at 4 °C for 3 hours. Post ChIP DNA was purified by Qiagen MinElute PCR Purification Kit, typically yield 2-11ng. HiChIP libraries were prepared using Kapa HyperPrep Kit (Roche, KK8502). 15 μl of Dynabeads MyOne Streptavidin T1 (Thermo Fisher, 65602) were used for biotin pull down. After end repair and A-tailing, 5 μl of Illumina TruSeq DNA UD Indexes were used for adaptor ligation at 20°C for 15min. Libraries were amplified for 12 to 13 PCR cycles (DNA>5ng: 12 cycles, DNA<5ng: 13 cycles). NextSeq 500 SRX15052675 GSM6080945_r1 SRP372884 PRJNA833451 SRS12798365 GSM6080945 GSM6080945 OTHER GENOMIC other 10 million SEM Cells were resuspended in 10mL fresh cell media, and crosslinked by 2% formaldhyde for 10 min with rotating at room temperature. Crosslinking were quenched by glycine to a final concentration of 0.2M and incubating for 5 min with rotating at room temperature. Cells were washed once by 1 mL of cold PBS, flash-froze cell pellets in dry ice/ethanol bath and store at -80°C HIChIP was performed as previously described in (Mumbach et. al. 2017) with the following modifications. 15 μl of 25U/μl MboI restriction enzyme (New England Biolabs, R0147M) was used for digesting chromatin from 10 million cells at 37°C for 2 hours with shaking. Proximity ligation was performed overnight at 16°C on Eppendorf thermomixer with 900rpm shaking speed. Post proximity ligated products were washed once by 1 mL of Covaris shearing buffer (10mM Tris-HCl pH8.0, 1mM EDTA, 0.1% SDS, 1x cOmplete EDTA-free protease inhibitor) and resuspended in 1mL Covaris shearing buffer then transfer to Covaris milliTUBE. Sonication was performed with Covaris LE220-plus with the following settings: Duty Cycle = 30%, PIP = 400, Cycles/Burst = 200, Dithering = x +- 0.5mm, Y +- 0.5mm, z +- 0.0mm, speed 10mm/s, pause duration 0.0 and time = 10min. 120 μl DiaMag protein G beads (diagenode, C03010021) were pre-blocked in ChIP dilution buffer plus 0.1% BSA overnight at 4°C. After Covaris shearing, add 3 mL ChIP dilution buffer plus protease inhibitor to 1 mL of sheared chromatin, then precleared with 60ul pre-blocked DiaMag protein G beads at 4°C for 1 hour. 8 μg of Diagenode CTCF antibody (C15410210-50, 2.3 μg/μl) was used for 10 million cells and rotated at 4 °C overnight. Another pre-blocked 60 μl DiaMag protein G beads were added next day and incubate at 4 °C for 3 hours. Post ChIP DNA was purified by Qiagen MinElute PCR Purification Kit, typically yield 2-11ng. HiChIP libraries were prepared using Kapa HyperPrep Kit (Roche, KK8502). 15 μl of Dynabeads MyOne Streptavidin T1 (Thermo Fisher, 65602) were used for biotin pull down. After end repair and A-tailing, 5 μl of Illumina TruSeq DNA UD Indexes were used for adaptor ligation at 20°C for 15min. Libraries were amplified for 12 to 13 PCR cycles (DNA>5ng: 12 cycles, DNA<5ng: 13 cycles). NextSeq 500