GSM7676789: Spic-ChIP-seq-OE12-2iLd25_2; Mus musculus; ChIP-Seq
mESCs were cultured in SL or 2iL medium for 3 days. For each replicate, 15 million cells were fixed with 1% paraformaldehyde. Fixed cells were consecutively treated with buffers A (148mM NaCl, 1.48 mM EDTA, 0.74 mM EGTA, 74mM HEPES), Buffer-B (10 mM EDTA, 0.5 mM EGTA, 20 mM HEPES, 0.25% Triton-x100), and Buffer-C (150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 50 mM HEPES). Isolated nuclei were lysed in freshly made sonication buffer (150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 20 mM HEPES, 0.15% SDS, 1% TritonX-100, 1X protease inhibitor) and samples were sonicated using Biorapture Pico (Diagenode). Sonicated chromatin was precleared using BSA blocked protein A/G beads (Invitrogen), and was further incubated with 10 µg of primary antibody (anti-FLAG from Sigma for NANOG ChIP, and anti-GFP from Abcam for SPIC-GFP ChIP). After overnight incubation, the immune complexes were captured by incubation with BSA blocked protein A/G beads for 2h on a rotating wheel at 4ºC. Beads were washed with ChIP wash buffer 1 (2 mM EDTA, 20 mM Tris pH:8 150 mM NaCl, 1% TritonX-100, 0.1% SDS), Buffer-2 (2 mM EDTA, 20 mM Tris pH:8 150 mM NaCl, 1% TritonX-100, 0.1% SDS, 500 mM NaCl) and Buffer-3 (1 mM EDTA, 10 mM Tris pH:8) for 10 minutes. Samples were then incubated with fresh ChIP elution buffer (0.1 M NaHCO3, 1% SDS) for 30 min at RT. Eluted chromatin was de-crosslinked overnight by adding 5M NaCl and proteinase K (final 10mg/ml) and incubating at 65ºC. DNA was purified using the MinElute PCR Purification Kit (Qiagen) ChIP-Rx-seq libraries were subjected to library preparation using the KAPA HyperPrep kit (Roche) according to manufacturer's instructions with dual indices and sequencing on an Illumina HiSeq 2500 sequencer (dual-indexed 50-bp paired-end reads).