SRX17982147 GSM6665370_r1 SRP403887 PRJNA892898 SRS15496646 GSM6665370 GSM6665370 OTHER GENOMIC other Human tonsil tissue was processed by mincing with scissors followed by transfer of up to 4g of tissue to a gentleMACS C tube (Miltenyi Biotec) containing 8 mL of phosphate-buffered saline (PBS) with 0.5 mg/mL collagenase D and 3000 U/mL DNaseI, then dissociated on a GentleMACS Octo Dissociator (Miltenyi Biotec) using “program C (Spleen program 2 followed by spleen program 3).” Tissue homogenates were incubated in a 37°C water bath for 15 minutes, then dissociated again using “program C” and transferred through a 100 μm cell strainer into 20mL RPMI containing 10% human AB serum (Sigma Aldrich). Next, cell suspension was overlaid on 10mL of Ficoll-Paque PLUS (GE Healthcare) and subjected to density-gradient separation via centrifugation for 20 min at 1800rpm, 20°C, slow acceleration, no brake. Leukocytes were collected from the interphase layer and then washed with 50mL of PBS for 6 minutes at 1600rpm for 7 minutes, 20°C. Single cell suspensions of tonsil mononuclear cells were subjected to positive selection with anti-human-CD3, anti-human-CD19 and anti-human-CD14 (Miltenyi Biotec) and transferred through LD columns (Miltenyi Biotec) according to the manufacturer guidelines. The depleted cell suspension flowthrough was collected into 15mL conical and then centrifuged for 5 minutes at 1200rpm, 20°C. Subsequently, cells were labeled with LIVE/DEAD™ Fixable Near-IR dead cell stain kit (Invitrogen). Next, cells were labeled with sorting antibody cocktail which contained negative linage (Lin-) CD19 Brilliant Violet (BV)421 (HIB19), CD14-BV421 (63D3) and CD3-BV421 (OKT3), and the following antibodies: CD45- FITC, (HI30), CD94-PerCP-Cy5.5 (DX22), CD127-PE-Cy7 (A019D5), cKit-BV510 (104D2) and NKp44-Alexa Fluor (AF)647 (P44-8) all purchased from Biolegend (San Diego, CA), CRTH2-PE (301109, R&D). ILC3 cells were sorted based on the expression of CD45+Lin-CD127+CD94-CRTH2-cKit+NKp44+, similarly to Bar-Ephraim et al. Cell sorting was performed using a FACSAria II sorter (BD Biosciences, Mountain View, CA, USA). Post sorting sorted ILC3 cells were washed with PBS for 5 minutes at 1200rpm, 20°C and then incubated in 100L of 2% formaldehyde (in PBS) for 10 minutes, followed by an addition of 0.125M glycine. Next, cells were centrifuged at 400g for 5 minutes at 4°C, resuspended with cold PBS and centrifuged again at 400g for 5 minutes at 4°C, supernatant was discarded, and cells were snap frozen in liquid nitrogen and then stored and -80°C prior to PCHi-C analysis. Promoter Capture Hi-C was performed as previously described (doi: 10.1016/j.cell.2021.03.051). Cells were fixed in 2% PFA for 10 minutes, lysed in a lysis buffer (30 minutes on ice), and digested with DpnII (NEB) overnight at 37C rotating (950rpm). Restriction overhangs were filled-in with Klenow (NEB) using biotin-14-dATP (Jena Bioscience), and ligation was performed in the ligation buffer for 4 hours at 16C (T4 DNA ligase; Life Technologies). After overnight decrosslinking at 65C, the ligated DNA was tagmented to produce fragments of 300-700 bp range. Ligation products were isolated using MyOne C1 streptavidin beads (Life Technologies), followed by washing with Wash&Binding buffer and nuclease-free water. Isolated Hi-C ligation products on the beads were then used directly for PCR amplification, and the final Hi-C library was purified with AMPure XP beads (Beckman Coulter). Promoter Capture Hi-C was performed using a custom-design Agilent SureSelect system following the manufacturer's protocol. Illumina HiSeq 2500 SRX17982148 GSM6665371_r1 SRP403887 PRJNA892898 SRS15496648 GSM6665371 GSM6665371 OTHER GENOMIC other Human tonsil tissue was processed by mincing with scissors followed by transfer of up to 4g of tissue to a gentleMACS C tube (Miltenyi Biotec) containing 8 mL of phosphate-buffered saline (PBS) with 0.5 mg/mL collagenase D and 3000 U/mL DNaseI, then dissociated on a GentleMACS Octo Dissociator (Miltenyi Biotec) using “program C (Spleen program 2 followed by spleen program 3).” Tissue homogenates were incubated in a 37°C water bath for 15 minutes, then dissociated again using “program C” and transferred through a 100 μm cell strainer into 20mL RPMI containing 10% human AB serum (Sigma Aldrich). Next, cell suspension was overlaid on 10mL of Ficoll-Paque PLUS (GE Healthcare) and subjected to density-gradient separation via centrifugation for 20 min at 1800rpm, 20°C, slow acceleration, no brake. Leukocytes were collected from the interphase layer and then washed with 50mL of PBS for 6 minutes at 1600rpm for 7 minutes, 20°C. Single cell suspensions of tonsil mononuclear cells were subjected to positive selection with anti-human-CD3, anti-human-CD19 and anti-human-CD14 (Miltenyi Biotec) and transferred through LD columns (Miltenyi Biotec) according to the manufacturer guidelines. The depleted cell suspension flowthrough was collected into 15mL conical and then centrifuged for 5 minutes at 1200rpm, 20°C. Subsequently, cells were labeled with LIVE/DEAD™ Fixable Near-IR dead cell stain kit (Invitrogen). Next, cells were labeled with sorting antibody cocktail which contained negative linage (Lin-) CD19 Brilliant Violet (BV)421 (HIB19), CD14-BV421 (63D3) and CD3-BV421 (OKT3), and the following antibodies: CD45- FITC, (HI30), CD94-PerCP-Cy5.5 (DX22), CD127-PE-Cy7 (A019D5), cKit-BV510 (104D2) and NKp44-Alexa Fluor (AF)647 (P44-8) all purchased from Biolegend (San Diego, CA), CRTH2-PE (301109, R&D). ILC3 cells were sorted based on the expression of CD45+Lin-CD127+CD94-CRTH2-cKit+NKp44+, similarly to Bar-Ephraim et al. Cell sorting was performed using a FACSAria II sorter (BD Biosciences, Mountain View, CA, USA). Post sorting sorted ILC3 cells were washed with PBS for 5 minutes at 1200rpm, 20°C and then incubated in 100L of 2% formaldehyde (in PBS) for 10 minutes, followed by an addition of 0.125M glycine. Next, cells were centrifuged at 400g for 5 minutes at 4°C, resuspended with cold PBS and centrifuged again at 400g for 5 minutes at 4°C, supernatant was discarded, and cells were snap frozen in liquid nitrogen and then stored and -80°C prior to PCHi-C analysis. Promoter Capture Hi-C was performed as previously described (doi: 10.1016/j.cell.2021.03.051). Cells were fixed in 2% PFA for 10 minutes, lysed in a lysis buffer (30 minutes on ice), and digested with DpnII (NEB) overnight at 37C rotating (950rpm). Restriction overhangs were filled-in with Klenow (NEB) using biotin-14-dATP (Jena Bioscience), and ligation was performed in the ligation buffer for 4 hours at 16C (T4 DNA ligase; Life Technologies). After overnight decrosslinking at 65C, the ligated DNA was tagmented to produce fragments of 300-700 bp range. Ligation products were isolated using MyOne C1 streptavidin beads (Life Technologies), followed by washing with Wash&Binding buffer and nuclease-free water. Isolated Hi-C ligation products on the beads were then used directly for PCR amplification, and the final Hi-C library was purified with AMPure XP beads (Beckman Coulter). Promoter Capture Hi-C was performed using a custom-design Agilent SureSelect system following the manufacturer's protocol. Illumina HiSeq 2500 SRX17982149 GSM6665372_r1 SRP403887 PRJNA892898 SRS15496647 GSM6665372 GSM6665372 OTHER GENOMIC other Human tonsil tissue was processed by mincing with scissors followed by transfer of up to 4g of tissue to a gentleMACS C tube (Miltenyi Biotec) containing 8 mL of phosphate-buffered saline (PBS) with 0.5 mg/mL collagenase D and 3000 U/mL DNaseI, then dissociated on a GentleMACS Octo Dissociator (Miltenyi Biotec) using “program C (Spleen program 2 followed by spleen program 3).” Tissue homogenates were incubated in a 37°C water bath for 15 minutes, then dissociated again using “program C” and transferred through a 100 μm cell strainer into 20mL RPMI containing 10% human AB serum (Sigma Aldrich). Next, cell suspension was overlaid on 10mL of Ficoll-Paque PLUS (GE Healthcare) and subjected to density-gradient separation via centrifugation for 20 min at 1800rpm, 20°C, slow acceleration, no brake. Leukocytes were collected from the interphase layer and then washed with 50mL of PBS for 6 minutes at 1600rpm for 7 minutes, 20°C. Single cell suspensions of tonsil mononuclear cells were subjected to positive selection with anti-human-CD3, anti-human-CD19 and anti-human-CD14 (Miltenyi Biotec) and transferred through LD columns (Miltenyi Biotec) according to the manufacturer guidelines. The depleted cell suspension flowthrough was collected into 15mL conical and then centrifuged for 5 minutes at 1200rpm, 20°C. Subsequently, cells were labeled with LIVE/DEAD™ Fixable Near-IR dead cell stain kit (Invitrogen). Next, cells were labeled with sorting antibody cocktail which contained negative linage (Lin-) CD19 Brilliant Violet (BV)421 (HIB19), CD14-BV421 (63D3) and CD3-BV421 (OKT3), and the following antibodies: CD45- FITC, (HI30), CD94-PerCP-Cy5.5 (DX22), CD127-PE-Cy7 (A019D5), cKit-BV510 (104D2) and NKp44-Alexa Fluor (AF)647 (P44-8) all purchased from Biolegend (San Diego, CA), CRTH2-PE (301109, R&D). ILC3 cells were sorted based on the expression of CD45+Lin-CD127+CD94-CRTH2-cKit+NKp44+, similarly to Bar-Ephraim et al. Cell sorting was performed using a FACSAria II sorter (BD Biosciences, Mountain View, CA, USA). Post sorting sorted ILC3 cells were washed with PBS for 5 minutes at 1200rpm, 20°C and then incubated in 100L of 2% formaldehyde (in PBS) for 10 minutes, followed by an addition of 0.125M glycine. Next, cells were centrifuged at 400g for 5 minutes at 4°C, resuspended with cold PBS and centrifuged again at 400g for 5 minutes at 4°C, supernatant was discarded, and cells were snap frozen in liquid nitrogen and then stored and -80°C prior to PCHi-C analysis. Promoter Capture Hi-C was performed as previously described (doi: 10.1016/j.cell.2021.03.051). Cells were fixed in 2% PFA for 10 minutes, lysed in a lysis buffer (30 minutes on ice), and digested with DpnII (NEB) overnight at 37C rotating (950rpm). Restriction overhangs were filled-in with Klenow (NEB) using biotin-14-dATP (Jena Bioscience), and ligation was performed in the ligation buffer for 4 hours at 16C (T4 DNA ligase; Life Technologies). After overnight decrosslinking at 65C, the ligated DNA was tagmented to produce fragments of 300-700 bp range. Ligation products were isolated using MyOne C1 streptavidin beads (Life Technologies), followed by washing with Wash&Binding buffer and nuclease-free water. Isolated Hi-C ligation products on the beads were then used directly for PCR amplification, and the final Hi-C library was purified with AMPure XP beads (Beckman Coulter). Promoter Capture Hi-C was performed using a custom-design Agilent SureSelect system following the manufacturer's protocol. Illumina HiSeq 2500