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. DNA samples were prepared according to TurSeq ChIP sample preparation guide (Illumina)
Experimental Factor: No IP (whole cell extract)
immunoprecipitate
Experimental Factor: H3K9M mutant
genotype