title: GSM4678108: pMIG input_rep2 ChIPseq; Mus musculus; ChIP-Seq
library_construction_protocol: ChiP was performed as described below. pMIG-, BATF- transduced CD8+ T cells (1x106 cells/ml in culture media) were fixed with 1% formaldehyde at room temperature for 10 minutes with nutation. To quench the fixation, 0.5 ml 2.5M glycine was added per 10 ml, the cells were incubated on ice for 5 minutes, and washed twice with cold PBS. Fixed cells were transferred to low-binding tubes with 1 ml cold PBS and spun down at 2000rpm at 4℃ for 10 min. Cells were pelleted, snap-frozen with liquid nitrogen, and stored at -80℃ until further processing. To isolate nuclei, cell pellets were thawed on ice and the pellets were resuspended in 1 ml Bioruptor lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% NP40, 0.25% Triton X-100), and incubated for 10 min at 4℃ with nutation. After centrifugation at 1700g at 4℃ for 5min, the resulting nuclear pellets were washed twice with washing buffer (10 mM Tris-HCl pH8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA). The pellets were resuspended in 100 l shearing buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA. 1% SDS), and sonicated using a Bioruptor in 1.5 ml bioruptor tubes (10 cycles, 30 seconds on, 30 seconds off). After sonication, the supernatants were transferred to 1.5 ml lo-binding tubes, and insoluble debris was removed by centrifugation at 20,000g. Pellets were resuspended in 100 µl shearing buffer, and 9 volumes of conversion buffer (10 mM Tris-HCl pH 7.5, 255 mM NaCl, 1 mM EDTA, 0.55 mM EGTA. 0.11% Na Deoxycholate, 0.11 % Triton X-100) was added. Chromatin was precleared with washed protein A and protein G Dynabeads for 1 hour, and the chromatin concentration was measured by qubit. 5% of chromatin was saved as input, and chromatin was incubated with anti-BATF (Brookwood Biomedical, USA) or anti-IRF4 (clone D9P5H, Cell Signaling Technology, USA) antibodies and protein A and protein G Dynabeads overnight at 4℃ with rotation. The following day, bead-bound chromatin was washed twice with RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.1% SDS, 0.5% Na Deoxycholate), and then with high salt buffer (50 mM Tris-HCl pH8.0, 500 mM NaCl, 1 mM EDTA, 1% NP40, 0.1% SDS), LiCl buffer (50 mM Tris-HCl pH 8.0, 250 mM LiCl, 1 mM EDTA, 1% NP40, 1% Na Deoxycholate), and TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Chromatin was eluted with 100 l elution buffer (100 mM NaHCO3, 1% SDS, 1 mg/ml RNase A) twice for 30 minutes at 37℃ using a 1000 rpm shaking heat block. 5 l proteinase K (20 mg/ml, Ambion, USA) and 8 l of 5 M NaCl were added to the eluted DNA, and samples were incubated at 65℃ with shaking (1,200 rpm) for de-crosslinking. DNA was purified with Zymo ChIP DNA Clean & Concentrator (Zymo Research, USA). Libraries were prepared using NEB Ultra II library Prep kits (NEB) following the manufacturer's instructions ChiP-seq, 2x50- cycle paired-end sequencing (illumina)
attributes.tag: GEO Accession
attributes.value: GSM4678108