title: GSM4066081: D3_V5_ChIPSeq; Mus musculus; ChIP-Seq
library_construction_protocol: Cell pellets were thawed in ice and resuspended in cell lysis buffer (5 mM PIPES, pH 8.0, 85 mM KCl, and 0.5% NP-40, 1 ml/15 cm plate) and incubated for 10 min on ice. Lysates were then centrifuged for 10 min at 4000 rpm. Nuclear pellets were resuspended in 6 volumes of sonication buffer (50 mM Tris-HCl, pH 8.1, 10 mM EDTA, 0.1% SDS), incubated on ice for 10 min, and sonicated to obtain DNA fragments below 2000 bp in length (Covaris S220 sonicator, 20% Duty factor, 200 cycles/burst, 100 peak incident power, 32 cycles of 20" on and 40" off).  Sonicated lysates were cleared by centrifugation and 800 μg of chromatin was diluted in RIPA buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate, 140 mM NaCl) to a final concentration of 0.8 μg/μl, precleared with Protein A sepharose (GE Healthcare) for 2 hours at 4°C and immunoprecipitated overnight with 8 μg of antibodies. 4% of the precleared chromatin was saved as input.  Immunoprecipitated DNA was purified with the Qiagen QIAquick PCR Purification Kit, eluted in 60 μl of water and used for library preparation. Libraries were prepared with the Illumina ChIP-seq DNA sample preparation kit according to manufacturer instructions (for input DNA we used only 150 ng of DNA)
attributes.tag: GEO Accession
attributes.value: GSM4066081