GSM1654727: Gcn5_1_ChIPSeq; Mus musculus; ChIP-Seq
Approximately 2.0 x 107 BirAV5-FLBioGcn5 and BirAV5-FLBio mESCs were crosslinked with 1% formaldehyde in the culture media for 10 min at room temperature, quenched with 125 mM glycine and washed three times with PBS containing protease inhibitors. The cells were lysed in SDS Lysis Buffer (1 % SDS, 10 mM EDTA and 50 mM Tris-HCl, pH 8.1) for 30 min on ice and sonicated with a Branson Sonifier 450 sonicator (output 1.5, duty 60 %) for 2 cycles, followed by 10 min in the Bioruptor (Diagenode) for 30 sec on/off at the high setting for 5 cycles. Sonicated samples were centrifuged to remove the insoluable debris, diluted 1:10 with ChIP Dilution Buffer (0.01 % SDS, 1.1 % Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1 and 167 mM NaCl) and were precleared with Dynabeads Protein A (Invitrogen) for 1.5 hrs. Subsequently, the pre-cleared lysates were incubated with Dynabeads MyOne Streptavidin T1 (Invitrogen) overnight at 4oC. The subsequent bioChIP steps were carried out as previously described (Kim et al. 2009). Libraries were prepared using a modified version of the Illumina TruSeq ChIP Sample Prep protocol. Briefly, ChIP DNA was end repaired (End-It DNA End-Repair Kit - Epicenter), 3’ adenylated with Klenow 3’-5’ exo (New England Biolabs) and ligated to Illumina Tru-Seq adapters (diluted 1:7) with DNA ligase (New England Biolabs). 5 x PCR amplification cycles were performed on the adapter ligated ChIP DNA, 250-300 bp products were purified from a 2 % agarose gel and amplified for 10 additional PCR cycles. Libraries concentrations were determined with a Library Quantification Kit (Kapa Biosystems). 10 pM of each library was sequenced on an Illumina HiSeq 2000 as outlined by Illumina.
GEO Accession
GSM1654727