GSM4633458: H3K27ac chip-seq in Ubc9-depleted mouse ESCs, replicate 1; Mus musculus; ChIP-Seq
ChIP-seq was done based on how it was described earlier in PMID: 24048479. After harvesting, washing and centrifugate, the cell pellets were crosslinked in 1% formaldehyde for 10 minutes at room temperature, and then quenched with 2.5 M Glycine for 5 minutes at room temperature. Fixated cells were then lysed in a Lysis buffer (140 mM NaCl, 50 mM HEPES KOH pH 7.5, 10% glycerol, 0.5% NP-40 alternative, 0.25% Triton X-100, 1 mM EDTA, supplemented with a complete protease inhibitor cocktail tablet (Roche) and 20 mM NEM) at 4°C. The lysates were then centrifuged at 1000g for 10 minutes at 4°C, and the pellet was resuspended in a Sonication Solution (140 mM NaCl, 10 mM Tris HCl, 0.1% DOC, 0.2% SDS and complete protease inhibitor cocktail and 20 mM NEM) and were sonicated with Sonifier (model S-450D) at 4°C  to achieve size ranges between 200 and 800 bp and then precipitated at full speed for 10 minutes by a cooled centrifuge. Supernatant lysates were extracted and diluted. Small amount of lysate was saved for whole cell extract (input) at this point. Antibodies were pre-bound by incubating with Protein-G Dynabeads (Invitrogen) in blocking buffer (0.5% Tween and 0.5% BSA in PBS) for 1 hour at room temperature. Washed beads were added to the lysates for incubation, as detailed below per antibody. Following incubations, the samples were washed five times with the Lysis buffer, twice with modified Lysis buffer (500 mM NaCl, 50 mM HEPES KOH pH 7.5, 10% glycerol, 0.5% NP-40 alternative, 0.25% Triton X-100, 1 mM EDTA, supplemented with a complete protease inhibitor cocktail tablet (Roche), twice with LiCl buffer (0.5% NP-40, , 250 mM LiCl, 10 mM TE, 0.5% DOC), once with TE (10 mM Tris-HCl pH 8.0, 1mM EDTA), and then eluted in Elution buffer (0.5% SDS, 300 mM NaCl, 5 mM EDTA, 10 mM Tris HCl pH 8.0). Eluate were treated sequentially with RNaseA (Roche,) for 30 minutes in 37 °C and Proteinase K (NEB) for 2 hours at 37°C and were then de-crosslinked at 65 °C for 8 hours. DNA was purified with Agencourt AMPure XP system (Beckman Coulter Genomics). Libraries were then prepared according to a modified version of the Illumina Genomic DNA protocol, as detailed previously in PMID: 24048479. H3K27ac: 5 million cells, 5ug ab4729 (Abcam), incubation 6 hours incubation time at 4°C. SUMO1: 5 million cells, 5ug 4930 (Cell Signaling Technologies), incubation 12 hours incubation time at 4°C.
GEO Accession
GSM4633458