GSM4822360: BR172_notag_FlagChIP; Schizosaccharomyces pombe; ChIP-Seq
Log-phase yeast cells were incubated at 18 ̊C for 2 hours and then fixed for 30 minutes in 3% freshly made formaldehyde. The crosslinking reaction was stopped by the addition of 2.5 M Glycine to make a final concentration of 0.125 M. The cells were pelleted and washed with PBS (phosphate buffered saline) before resuspended in ChIP lysis buffer (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1% Triton X-100, 0.1% Deoxycholate supplemented with cOmplete protease inhibitors cocktail (Roche)). Ice cold glass beads were added and the mixtures were vigorously disrupted in a bead- beater. The lysates were collected and subjected to sonication to reduce chromatin size to 500– 1000 base pairs. The cleared cell lysates were incubated with anti-Flag (Sigma) over night at 4 ̊C. Protein G beads were then added to isolate the antibodies and associated chromatin fragments. The beads were then washed with ChIP lysis buffer twice, ChIP lysis buffer containing 0.5 M NaCl, Wash buffer (10 mM Tris, pH 8.0, 250 mM LiCl, 0.5% NP-40, 0.5% Deoxycholate, 1 mM EDTA), and TE (50 mM Tris pH 8.0, 1 mM EDTA). The bound chromatin fragments were eluted with TES (50 mM Tris pH 8.0, 1 mM EDTA, 1% SDS) and the crosslinking was reversed by incubating at 65 ̊C overnight. The protein DNA mixture were then subjected to proteinase K treatment and phenol:chloroform extraction before the DNA was precipi- tated by ethanol. Libraries were prepared according to TruSeq ChIP sample preparation guide (Illumina)