GSM1545026: Input_DNA (ChIP-Seq control); Mus musculus; ChIP-Seq
Mouse R1 embryonic stem cells (Nagy et al., 1993) (10^8 cells) were cross-linked in complete medium (10% FBS) containing 1% formaldehyde for 10 min, the reaction was terminated by addition of 125 mM glycine. Fixed cells were washed three times (5 min each) in cold PBS and lysed in LB1 buffer (LB2 buffer containing 0.5% NP-40 and 0.25% triton X-100). Nuclei were then washed in LB2 buffer (10mM Tris-HCl pH=8 and 200mM NaCl) to remove detergents and resuspended in LB3 buffer (LB2 buffer containing 0.1% Na-deoxycholate and 0.5% N-lauroylsarcosine). Chromatin was sonicated 5 times for 30 sec cycles at 30% of the maximum power of a Branson 450 sonicator to generate 100-400 bp chromatin fragments. After clearing by centrifugation, sonicated chromatin was incubated with antibody-bound protein A-conjugated magnetic beads (Invitrogen, Carlsbad, USA). For each IP we used 10 ug antibody. IP with rabbit IgG was performed as negative control. After overnight IP at 4°C, the bound complexes were washed twice in WB1 (50 mM Hepes-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton-X100, 0.1% Na-doexycholate), twice in WB2 (50 mM Hepes-KOH pH 7.5, 500 mM NaCl, 1 mM EDTA, 1% Triton-X100, 0.1% Na-doexycholate) and twice in LiCl WB (10 mM Tris-Cl pH 8.0, 250 mM LiCl, 0.5% NP-40, 0.5% Na-deoxycholate, 1 mM EDTA). Immunoprecipitated complexes were eluted from the beads by incubation for 30 min in EB (2% SDS in TE) at 37°C. The eluted material was reverse cross-linked at 65°C overnight and incubated for 1 h at 55°C with proteinase K. The obtained material was extracted with phenol-chloroform and ethanol precipitated. After RNAse treatment, the DNA was purified with a PCR purification kit (Qiagen, Netherlands). About 10 ng of immunoprecipitated DNA were processed for sequencing. The input and ChIP DNA were used for preparation of sequencing libraries according to the manufacturer’s instructions for the Genome Analyzer GAII instrument (Illumina). After DNA purification with a DNA Clean & Concentrator-5 kit (Zymo Research), the DNA libraries were separated on 2% agarose TAE gels. The 150- to 250-bp fragments were excised from the gel on a Dark Reader (Claire Chemical Research) and purified with a MinElute Gel Extraction Kit (Qiagen). Sequencing was performed on a Genome Analyzer GAII instrument (Illumina) with 36 cycles.