GSM4977090: I20-1022-03: WT_Day3_H3K27me3_Rep1; Mus musculus; ChIP-Seq
50.10^6 cells were trypsinised, resuspended and fixed in 50 mL warm medium and 1% PFA for 10 min at 37oC. 2.5 mL Glycine 2.5 M were added to the cells (final concentration 0.125 M) to quench the PFA, 5 min RT on rotator. All buffers from now on contain protease inhibitors (Roche, 4693132001). Cells were washed twice with cold PBS. Then 1x in 10 mL Buffer 1 (10 mM Hepes pH 7.5, 10 mM EDTA, 0.5 mM EGTA, 0.75% Triton X-100) and 1x in Buffer 2 (10 mM Hepes pH 7.5, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA), 10 min rotating in at 4oC. Nuclei were then resuspended in Lysis/Sonication Buffer (150 mM NaCl, 25 mM Tris pH 7.5, 5 mM EDTA, 1% Triton, 0.1% SDS, 0.5% Deoxycholate) and left 30 min on ice. Nuclei were then sonicated 2x 15 min (30'' ON/OFF, max input, ice cold water) in a Bioruptor. A small fraction of the lysate was run on a gel to confirm size population of 100-500 bp. 1 µg of antibody (Cell Signaling, 9733S) was conjugated with 25 µL magnetic beads (Life Technologies, 10004D) for 3h rotating at 4oC. 25 ug of chromatin was IP'd with the Ab-beads overnight rotating at 4oC. Beads were washed 2x in standard RIPA buffer (140 mM NaCl, 10 mM Tris-HCl pH 7.5150 mM NaCl, 1 mM EDTA pH8.0, 0.5 mM EGTA pH 8.0, 1% Triton, 0.1% SDS, 0.5% Deoxycholate), 1x in High Salt RIPA (same as standard RIPA but with 500 mM NaCl), 1x LiCl RIPA (same as standard RIPA but with 250 mM LiCl instead of NaCl) and rinsed once with TE, 10 min 4oC each wash. Chromatin was eluted with 450 µL of Elution Buffer (1% SDS; 0.1M NaHCO3 in Water) with 22 ul protease K (10 mg mL-1) and 5 ul RNAse A (10 mg mL-1) and shaken at 1000 rpm for 2hrs at 37oC first and then 65oC overnight. DNA was then Phenol-Chloroform extracted and resuspended in 20 µL H20. 0.25 µL was used per PCR to confirm the ChIP's success. The concentration was then measured. The ChIP-seq library was prepared following the manufacturer's instructions (ThruPLEX DNA, Takara Bio) and sequenced on an Illumina HiSeq 2500 sequencer (50 bp paired-end reads).