title: GSM8368587: D5-Teff-Rest-MED12-KO-Input_ChIP; Homo sapiens; ChIP-Seq
library_construction_protocol: A portion of edited Teffs were re-stimulated with ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator (STEMCELL Technologies, #10990) 10 days following isolation and collected 48 hours later. Up to 1-2 x 106 Teffs were cross-linked in PBS with 1% methanol-free formaldehyde (Thermo 28908) for 10 min at 18-22ºC followed by quenching in glycine at 125 mM final concentration. 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 P7626) and 0.5X protease inhibitor cocktail (PIC, Sigma P8340). Chromatin was sheared on a Covaris E220 focused ultrasonicator using 1 mL milliTubes (Covaris 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. Formaldehyde cross-linked, sheared mouse CD8 T cell chromatin was spiked in at 2.5% of human Teff chromatin based on fluorometric (Qubit, Thermo Q33238) or OD260 (Nanodrop, Thermo 912A1099) quantification. 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 (Supplemental table 21) bound to a 1:1 mixture of protein A and protein G magnetic beads (Thermo 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 E7370L) as described previously64. ChIP-seq libraries were multiplexed for paired-end (2 x 50bp) sequencing on an Illumina NextSeq 2000 instrument.