Schizosaccharomyces pombe 972h-
Protocols: 200 ml of cells were grown at 32C to a density of 1E7 cells/ml Cells were fixed by the addition of formaldehyde to a final concentration of 1%, followed by incubation for 30 minutes at 32°C. The reaction was stopped with 10 ml of 2.5 M glycine. Cells were washed extensively in phosphate-buffered saline solution (PBS), resuspended in lysis buffer (50 mM HEPES pH 7.6, 140 mM NaCl, 1 mM EDTA, 0.1% Triton X-100, 1 mM PMSF, and 1× complete protease inhibitor cocktail [Roche]), and lysed with a Fastprep 24 bead-beater (5 cycles of 20 seconds at level 6.5). Chromatin was sheared by sonication with a Bioruptor (Diagenode; 6 cycles of 5 minutes with 30 seconds on / 30 seconds off at high intensity). The lysates were cleared by centrifugation at 20,000 g for 10 minutes, and adjusted to 500 μl at 6 μg/μl total protein, as determined by the BCA assay. 100 µl of magnetic beads (Pan Mouse IgG or protein G, Life Technologies) were blocked with bovine serum albumin (BSA), and coated with 5 µg of mouse monoclonal antibodies against methylated H3K9 (H3K9me2, Abcam ab1120) or with anti-histone H3 rabbit polyclonal antibodies (Abcam ab1791), respectively. The extracts were incubated overnight with the coated beads at 4°C with rotation. Next, the beads were washed twice with 0.8 ml of 50 mM HEPES pH 7.6, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and 0.1% sodium deoxycholate, twice in the same buffer containing 500 mM NaCl, twice in 10 mM Tris-HCl pH 8, 250 mM LiCl, 1 mM EDTA, 0.5% NP-40, and 0.5% sodium deoxycholate, and once in Tris-EDTA solution. Elution and crosslinking reversal were performed by incubating the beads in 200 μl of 10 mM Tris-HCl pH 8, 1 mM EDTA, and 1% SDS overnight at 65°C. DNA was purified with Qiagen's a MinElute PCR purification kit (Qiagen) following the manufacturer's protocol, and eluted in 10 μl. Libraries were prepared using the Next ChIP-seq Library Prep Master Mix Set for Illumina (New England Biolabs) according to the manufacturer's instructions
ENA first public
2015-09-28
ENA last update
2014-11-03
External Id
SAMEA3108283
INSDC center alias
Department of Biochemistry University of Cambridge
INSDC center name
Department of Biochemistry University of Cambridge
INSDC first public
2015-09-28T17:01:13Z
INSDC last update
2014-11-03T11:10:32Z
INSDC status
public
Submitter Id
E-MTAB-3036:WT_H3_1
broker name
ArrayExpress
genotype
wild type genotype
sample name
E-MTAB-3036:WT_H3_1