GSM2359818: ZF_E_biological_rep1; Mus musculus; ChIP-Seq
5 x 10^5 MEFs were seeded onto a gelatin-coated 10 cm dish and infected with retrovial vectors the next day. 24 hours after infection, medium was replaced into KSR medium, and 48 hours after infection, cells were fixed with 1% formaldehyde in DMEM at room temperature for 5 min and quenched with glycine. Fixed cells were washed with PBS twice and treated with 0.5% NP40, 10 mM NaCl and 10 mM Tris-HCl for 10 min. Cells were scraped and collected into a tube and centrifuged at 3,000 rpm for 20 min. The cell pellet was lysed in 1% SDS, 10 mM EDTA, 1x cOmplete protease inhibitor (Roche) and 50 mM Tris-HCl, and then diluted one fourth with IP dilution buffer (50 mM Tris-HCl, 167 mM NaCl, 1.1% Triton X-100, 0.11% deoxycholate, 1x cOmplete). Cell lysates were sonicated with handy sonicator XL-2000 (Misonix) at level 7 for 10 sec 6 times and stored at -80 °C until the immunoprecipitation procedure. Before immunoprecipitation, a complex of 24 uL Dynabeads ProteinG (LifeTechnologies) and 2.4 ug anti-FLAG M2 antibody (Sigma) per 10 cm dish was formed in RIPA buffer [50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% TritonX-100, 0.1% deoxycholate, 1x Protease inhibitor (Nacalai Tesque, 25954-21)] by rotating at 4 °C, O/N. Antibody-bead complex was washed with RIPA buffer twice, then combined with sonicated cell lysates and rotated at 4 °C, O/N.  The resultant complexes were successively washed with RIPA, high-salt RIPA (RIPA with 500 mM NaCl), LiCl wash buffer (10 mM Tris-HCl, 250 mM LiCl, 1 mM EDTA, 0.5% NP40, 0.5% deoxycholate), TE buffer, and eluted twice with 100 mM NaHCO3, 1% SDS and 10 mM DTT. Eluates were supplemented with 200 mM final NaCl, de-crosslinked by shaking at 65 °C, O/N and treated with 50 ug/mL RNase A at 37 °C for 1 hr and 2 mg/mL Pronase at 42 °C for 2 hr. DNA was purified with PCR Purification Kit (Qiagen). Purified ChIP DNA (5 ng per sample) was used for end repair, 3’ end adenylation and adaptor ligation reaction with TruSeq ChIP Sample Preparation Kit (illumina) following the manufacturer’s manual. Resultant adaptor-ligated DNA was amplified by PCR, and subsequently selected for 300-450 bp fragments by gel electrophoresis followed by excision and extraction with Gel Extraction Kit (Qiagen). The resultant library DNA was quantified with Library Quantification Kit (KAPA Biosystems), and 100 mL of total 10 nM of mixture of indexed DNA libraries were used for single-end 100-bp sequencing by HiSeq 2000 (illumina).
GEO Accession
GSM2359818