GSM2359610: wt 1h, input for sample 26, rep1 (ChIP-Seq); Schizosaccharomyces pombe; ChIP-Seq
Equal number of cells at each time point was cross-linked with 1% formaldehyde at room temperature for 15min. Crosslinking was quenched with 0.125M glycine for 5min and the cells were washed 3 times with cold TBS. Cell pellets (about 2x109 cells) were resuspended in 400μl lysis buffer (20mM Hepes, 0.5M NaCl, 1mM EDTA, 1% Triton-X, 0.1% Na-Deoxycholate, 0.1% SDS, 1mM PMSF and 5mM Benzamidine) and lysed with glass beads. Whole cell extracts (WCEs) were sonicated for 10min using the Q-Sonica waterbath sonicator with final energy output of roughly 16000J producing chromatin 100-400bps in size. 2-4ug of antibody (abcam 1220 for H3K9me2 and active motif 61013 for H3K9me3) plus 10μl Protein A Dynabeads were added to each extract and incubated for 3 hours at 4 deg. Beads were then washed and eluted with TE plus 0.67% SDS at 65ºC for 15min. Samples were incubated at 65 ºC for 8 hours to reverse the crosslinks and then treated with RNaseA and proteinase K to remove RNA and proteins, respectively. Finally, DNA fragments were isolated by Phenol-Chloroform extraction followed by and ethanol precipitation. Libraries were made with NEBNext Ultra DNA Library rep Kit for Illumina (NEB E7370L)
GEO Accession
GSM2359610