GSM1479734: H3K27ac ZT 18 Bmal1 KO; Mus musculus; ChIP-Seq
ChIP-seq H3K27ac protocol: ChIPs were performed according to the method described by Reddy et al. (2009) with a few modifications. The 100 uL chromatin aliquots were used for each IP and diluted with 900 ul of RIPA buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS in PBS at pH 7.4) and added to dynal magnetic beads conjugated with (sheep anti-mouse IgG Dynabeads, Invitrogen, Cat no: 110-31) pre-treated with 3 ul of polyclonal antibody for H3K27ac (Active Motif, Cat no: 39135) for immunoprecipitation of specific complexes. The samples were incubated overnight at 4°C on rotator, then magnetic beads washed 7 times with lithiumchloride wash buffer (100mM Tris at pH 7.5, 500mM LiCl, 1% NP-40 and 1% sodiumdeoxycholate) and finally once with 1X TE buffer (10mM Tris-HCl at pH 7.5, 0.1mM Na2EDTA). The chromatin complex was eluted using elution buffer (1% SDS, 0.1M NaHCO3) for 1 h at 65°C using eppendorf thermo-mixer. The chromatin was then de-crosslinked overnight at 65°C and ChIP DNA purified using Qiagen PCR purification kit and eluted in 50 ul of elution buffer. For qPCR reaction, 1.5 ul of 1/10 diluted ChIP DNA is used. Libraries for ultra-high-throughput sequencing were prepared with the ChIP-Seq DNA sample kit (Illumina) as recommended by the manufacturer.
GEO Accession
GSM1479734