GSM9177352: Human Treg stim, Donor 3, FOXP3 ChIP, DSG/formaldehyde fixation; Homo sapiens; ChIP-Seq
Up to 2.5E7 cells were cross-linked in PBS with 1% methanol-free formaldehyde (Thermo, catalog no. 28908) for 10 min at room temperature followed by quenching in glycine at 125 mM final concentration. Tconv and selected Treg samples were cross-linked in 2 mM disuccinimidyl glutarate (Thermo, catalog no. 20593) in PBS for 30 min at room temperature prior to formaldehyde treatment. Cross-linked cell pellets were snap-frozen in liquid nitrogen and stored at –80ºC. Nuclei were isolated from thawed, cross-linked cells via sequential lysis in LB1 (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% IGEPAL CA-360, and 0.25% Triton X-100), LB2 (10 mM Tris-HCl pH 8, 200 mM NaCl, 1 mM EDTA, and 0.5 mM EGTA), and LB3 (10 mM Tris-HCl pH 8, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% sodium deoxycholate [NaDOC], and 0.5% N-laurylsarcosine) supplemented with 0.5 mM phenylmethylsulfonyl fluoride (PMSF, Sigma, catalog no. P7626) and 0.5X protease inhibitor cocktail (PIC, Sigma, catalog no. P8340). Chromatin was sheared on a Covaris E220 focused ultrasonicator using 1 mL milliTubes (Covaris, catalog no. 520128) with 140W peak incident power, 5% duty factor, 200 cycles per burst, 6ºC temperature setpoint (minimum 3ºC, maximum 9ºC), fill level 10, and time 12-14 min to obtain target size 200-700 bp. Triton X-100 was added to final concentration of 1% prior to immunoprecipitation for 16 h at 4ºC with 2-8 µg of indicated antibodies bound to a 1:1 mixture of protein A and protein G magnetic beads (Thermo, catalog nos. 10001D and 10003D). Bead-bound antibody-chromatin complexes were sequentially washed three times with Wash Buffer 1 (20 mM Tris pH 8, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium dodecyl sulfate [SDS], and 0.1% NaDOC), twice with Wash Buffer 2 (20 mM Tris-HCl pH 8, 500 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% SDS, and 0.1% NaDOC), twice with Wash Buffer 3 (20 mM Tris-HCl pH 8, 250 mM LiCl, 1 mM EDTA, 0.5% IGEPAL CA-360, and 0.5% NaDOC), twice with TET (10 mM Tris-HCl pH 8, 1 mM EDTA, 0.2% Tween-20), and once with TE0.1 (10 mM Tris-HCl pH 8, 0.1 mM EDTA, 0.5 mM PMSF, and 0.5X PIC) supplemented with 0.5 mM PMSF and 0.5X PIC. Beads were resuspended in TT (10 mM Tris-HCl pH 8, 0.05% Tween-20) prior to on-bead library preparation using the NEBNext Ultra II DNA Library Prep Kit (NEB, catalog no. E7370L) as described previously (Texari et al., 2021). ChIP-seq libraries were multiplexed for paired-end sequencing on an Illumina NextSeq 2000 or NovaSeq X instrument. On-bead library preparation was conducted using the NEBNext Ultra II DNA Library Prep Kit (NEB, catalog no. E7370L) as described previously (Texari et al., 2021). ChIP-seq libraries were multiplexed for paired-end sequencing.