GSM6254258: B cells, VC 4 days, pSTAT3, IL-21 6h, bio rep 1 [ChIP-seq]; Mus musculus; ChIP-Seq
Cells were fixed with 1% formaldehyde (ThermoFisher) in media at 1 × 106 cells/ml for 10 mins at room temperature with constant nutation. Fixation was quenched with 125 mM glycine for 5 min on ice. After being washed twice with PBS, cell pellets were snap-frozen with liquid nitrogen and stored at −80 °C until nuclei preparation. To extract chromatin, nuclei were isolated using lysis buffer (50 mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40, and 0.25% Triton X-100) and were washed once with wash buffer (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, and 0.5 mM EGTA), followed by two washes with shearing buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, and 0.1% SDS) without disturbing pellets. Pellets were then resuspended in shearing buffer and the chromatin was sheared using Bioruptor Pico (Diagenode) for 7 cycles (30 sec on, 30 sec off) at 4 °C. After precleared with ProteinA dynabeads (ThermoFisher), 20 μg of chromatin was diluted to a final of 500uL RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.1% SDS, and 0.5% sodium deoxycholate) and incubated with 1 μg rabbit monoclonal anti-phsopho-STAT3 (Tyr705) antibody (clone D3A7, Cell Signaling Technology) overnight at 4°C with constant rotation. To capture the antibody-bound chromatin, the samples were incubated with 30 μl of precleaned Protein A Dynabeads for 2 hours at 4 °C. The beads were washed twice with RIPA buffer and once with TE buffer (10 mM Tris-HCl pH 8.0 and 1 mM EDTA). All washes were incubated for 5 min at 4 °C with constant rotation. After the last wash, samples were eluted twice with 100 μl elution buffer (100 mM NaHCO3,1% SDS, and 0.5 mg/ml RNase A). To decrosslink, proteinase K (0.5 mg/ml; Ambion) and NaCl (200 mM) were added to the eluted samples, followed by the incubation at 65 °C overnight. The samples were purified using DNA Clean & Concentrator-5 (Zymo Research). Sequencing libraries were prepared using NEB Ultra II kits according to the standard protocol (NEB).