Illumina HiSeq 2500 paired end sequencing; ChIP-seq analysis of H3K9me3 levels across the genome for one copy of H3K9M mutant in pombe
ChIP-seq analysis of H3K9me3 levels across the genome for one copy of H3K9M mutant in pombe
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 2.5M Glycine to make a final concentration of 0.125M. The cells was pelleted and with PBS (phosphate buffered saline) before resuspended in ChIP lysis buffer (50 mM HEPES-KOH, pH7.5, 140 mM NaCl, 1% Triton X-100, 0.1% Deoxycholat supplemented with cOmplete protease inhibitors cocktail (Roche)). Ice cold glass beads were added and the mixtures were vigorously disrupted in a beadbeater. The lysates were collected and subjected to sonication to reduce chromatin size to 500-1000 base pairs. The protein DNA mixture were then subjected to proteinase K treatment and phenol:chloroform extraction before the DNA was precipitated by ethanol. The cleared cell lysates were incubated with antibodies: H3K9me3 (Abcam), H3K9me2 (Abcam), and 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 0.5M NaCl, Wash buffer (10 mM Tris, pH8.0, 250 mM LiCl, 0.5% NP-40, 0.5% Deoxycholate, 1 mM EDTA), and TE (50 mM Tris pH8.0, 1mM EDTA). The bound chromatin fragments were eluted with TES (50 mM Tris pH8.0, 1mM EDTA, 1% SDS) and the crosslinking was reversed by incubating at 65°C overnight.  DNA samples were prepared according to TurSeq ChIP sample preparation guide (Illumina)
Experimental Factor: IP by H3K9me3 antibody
immunoprecipitate
Experimental Factor: clr4 mutant
genotype