ChIPseq-H3K27me3_Input_2h
Cells were lysed in lysis buffer containing 1% SDS, 10 mM EDTA, 50 mM Tris pH 8.1 and protease inhibitors. Cells were sonicated for 60 cycles (7.5 min total sonication time) at low setting using a Bioruptor (Diagenode). Sonicated lysates were then cleared by centrifugation for 10 min at high speed, diluted 1/10 with dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris at pH 8.1, 0.1% Brij-35) and incubated with 10 l anti H3K27me3 (CST,C36B11) overnight at 4C. 50 l of Protein A Dynabeads (Life Technologies) were pre-incubated with 0.5% (w/v) BSA in PBS overnight. To capture antibody-bound protein-DNA complexes, lysates were incubated with the prepared beads for 4 hrs and subsequently washed twice with wash buffer I (0.1% SDS, 1% Triton X-100, 2 mM EDTA pH 8.0, 20 mM Tris pH 8.1, 150 mM NaCl), wash buffer II (0.1% SDS, 1% Triton X-100, 2 mM EDTA pH 8.0, 20 mM Tris pH 8.1, 500 mM NaCl), wash buffer III (0.25 mM LiCl, 1% NP-40, 1 % sodium deoxycholate, 1 mM EDTA pH 8.0, 10 mM Tris pH 8.1) and TE buffer (10 mM Tris pH 8.1, 1 mM EDTA pH 8.0) in the cold. DNA was eluted using 1% SDS, 100 mM NaHCO3 and reverse-crosslinked overnight before the clean up using AMpure XP beads