GSM8635213: ATF4_input_rep2; Mus musculus; ChIP-Seq
Treg cells (enriched CD4+CD25+ cells, 24 million per replicate) were cross-linked with 1% formaldehyde in PBS for 10 min at room temperature and the reaction was quenched by 0.125 M glycine. Cells were washed with ice-cold PBS, pelleted, snap-frozen in liquid nitrogen, and stored at −80 °C. On the day of ChIP, cells were thawed on ice and incubated in cell lysis buffer (10 mM HEPES-KOH pH 7.9, 1 mM EDTA, 85 mM KCl, 1% NP-40, 0.5 mM sodium butyrate, 1 mM PMSF, and 1x protease inhibitor cocktail) for 10 min. Nuclear pellets were resuspended in shearing buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5 mM EGTA, 1 mM EDTA, 0.1% SDS, 1%NP-40, 0.5 mM DTT, 0.4% sodium deoxycholate, 0.5 mM sodium butyrate, 1 mM PMSF, and 1x protease inhibitor cocktail) and sheared in a Covaris E220 sonicator. After centrifugation, the supernatant was diluted 1.1-fold with 10% Triton X-100 and 1% of the supernatant was kept as ChIP input. For immunoprecipitation, the supernatant was incubated with ATF4 antibody pre-bound to 20 ul of Protein A+G Dynabeads overnight at 4 °C. On the next day, the beads were collected and washed three times with WB I (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100, 0.5mM sodium butyrate, 1 mM PMSF, and 1x protease inhibitor cocktail), three times with WB III (10 mM Tris-HCl pH 7.5, 250 mM LiCl, 1 mM EDTA, 1%Triton X-100, 0.7% sodium deoxycholate, 0.5 mM sodium butyrate, 1 mM PMSF, and 1x protease inhibitor cocktail), and twice with TET (10 mM Tris-HCl pH 7.5, 0.2% Tween-20, 1 mM EDTA, 0.5 mM sodium butyrate, 1 mM PMSF, and 1x protease inhibitor cocktail). The beads were resuspended in 25 ul of TT (10mM Tris-HCl pH 7.5, 0.05% Tween-20) for on-bead library preparation by using NEBNext Ultra II DNA library prep kit (NEB) according to the manufacturer's protocol. DNA was eluted, crosslinks reversed by adding 4.5 ul of 5 M NaCl, 4 ul of 10% SDS, 3 ul of 0.5 M EDTA, 1 ul of 20 mg/ml proteinase K, and 20 ul of water, incubated at 55 °C for 1 h, and then incubated at 65 °C for 30 min. DNA was purified with 2 ul of SpeedBeads in 20% PEG 8000/1.5 M NaCl and eluted with 25 ul of TT. The eluted DNA was amplified and purified with 2 ul of SpeedBeads in 20% PEG 8000/2.5 M NaCl. The library was size selected using PAGE/TBE gel and sequenced on NovaSeq 6000. Libraries were prepared using NEBNext Ultra II DNA library prep kit (NEB) according to the manufacturer's protocol.