br910-clr4-ChIP
Schizosaccharomyces pombe
Protocols: 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)
ENA first public
2016-10-03
ENA last update
2016-09-29
External Id
SAMEA4471220
INSDC center alias
Columbia University in the City of New York
INSDC center name
Columbia University in the City of New York
INSDC first public
2016-10-03T17:01:37Z
INSDC last update
2016-09-29T12:15:17Z
INSDC status
public
Submitter Id
E-MTAB-4776:br910-clr4-ChIP
broker name
ArrayExpress
common name
fission yeast
genotype
clr4 mutant
sample name
E-MTAB-4776:br910-clr4-ChIP