GSM2045176: MEFs_input; Mus musculus; ChIP-Seq
Bmal1 ChIP in Smc3-flox/flox MEF cells were performed following the protocol by Shimomura et al. with modification. Briefly, 10e7 cells were washed with PBS and cross-linked by 1% formaldehyde for 10 min on a rocker at room temperature. The cross-linking was quenched by 2.5 M Glycine with final concentration of 125 mM. The nuclei was extracted at 4℃ from the homogenate by lysis buffer containing protease inhibitors [50mM Hepes-KOH, pH 7.5, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100], [10mM Tris-HCl, pH 8.0, 200mM NaCl, 1mM EDTA, 0.5 mM EGTA], and [10mM Tris-HCl, pH 8.0, 200mM NaCl, 1mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine]. DNA was fragmented with sonication into 150-300 bp at 4℃. 50 µl DNA fragments were stored in 4℃ as the input DNA. The rest of DNA fragments were incubated on rocker at 4℃ for 6 hr with 1:1 ChIP buffer [20% Triton, NaDOC, NaCl, TE, inhibitor] and 4 µl Bmal1 antibody (sc-8550). Then 15 µl protein A/G agarose beads were added into DNA and incubated on rocker at 4℃ overnight. Co-immunoprecipitated DNA was washed with 1 ml buffers [5% Triton, 1% SDS, 1% NaDOC, 93% TE] twice, [5% Triton, 1% SDS, 1% NaDOC, 6% NaCl, 87% TE] twice, [10% LiCl, 5% NP40, 5% Na-DOC, 80% TE] twice, [10% Triton, 90% TE], and TE. Then DNA was reverse cross-linked at 50℃ for 2 hr with TE 100 µl, 10% SDS 3 µl, and protease K 5 µl. QIAquick PCR Purification Kit (QIAGEN) was used to purified ChIP DNA. Input and ChIP DNA library were prepared by using Illumina TruSeq ChIP Sample Prep Kit and were subjected to deep sequencing with 100 bp read on HiSeq 2000 at CAS-MPG Partner Institute for Computational Biology Omics Core, Shanghai, China.
GEO Accession
GSM2045176