GSM9805157: Healthy human donor (3), CAR T cells, ATF3, ChIP, Rep1; Homo sapiens; ChIP-Seq
Up to 1e7 live CAR T cells (NGFR+) were enriched magnetically and cross-linked at RT first in 2 mM disuccinimidyl glutarate in PBS for 30 min and then in 1% methanol-free formaldehyde in PBS for 10 min. Formaldehyde was quenched by adding glycine to 125 mM final concentration. Cross-linked cells were pelleted, snap-frozen in liquid nitrogen, stored at –80ºC. Nuclei were isolated from cryopreserved pellet (1.5 x 1e6 cells) by sequential lysis in LB1 (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% IGEPAL CA-630, 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 and 0.5X protease inhibitor cocktail. Chromatin was fragmented to 200-700 bp on a Covaris E220 focused ultrasonicator in microTubes with the following parameters: 140W peak incident power, 5% duty factor, 200 cycles per burst, 6ºC temperature setpoint (range 3-9ºC), run level 6, and time 12-14 min. Triton X-100 was added to final concentration of 1% to clarified chromatin prior to immunoprecipitation for 16h at 4ºC with 2.5 uL of 1 mg/mL mouse monoclonal anti-FLAG M2 antibody bound to a 1:1 mixture of protein A and G magnetic beads. Immunoprecipitated, bead-bound chromatin was washed sequentially with the following buffers supplemented with 0.5 mM PMSF and 0.5X PIC: three times with WB1 (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 WB2 (20 mM Tris-HCl pH8, 500 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% SDS, and 0.1% NaDOC); twice with WB3 (20 mM Tris-HCl pH 8, 250 mM LiCl, 1 mM EDTA, 0.5% IGEPAL CA-630, 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). This mirrors the methods used previously (Umhoefer, et al., Immunity, 2025 and Arce, et al., Nature, 2025). Beads were brought up 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 as previously described (Texari, et al., STAR Protocols, 2021).