GSM3262717: vehicle treated, VDR IP, lane 1 of sequencing; Homo sapiens; ChIP-Seq
Immune complexes from immunoprecipitations of formalin-fixed, sonicated samples were collected with a 50% slurry of protein G for 1 h at 4oC. Cross-links were reversed overnight in 1% SDS, 0.1 M NaHCO3, and 0.2 M NaCl at 65oC.   DNA was purified using Qiagen QIAquick Spin Kits. We used methods originally reported in Robertson G. et al. (2007) Nature Methods 4:651-7.  Briefly, 100-300 bp fragments of the immunoprecipitated were eluted from 12%  PAGE gels and the DNA was recovered using a QIAquick PCR purification kit.  DNA ends were repaired using a 1:5 mix of T4 and Klenow DNA polymerases, phenol–chloroform–isoamyl alcohol (pH 8.0) extracted, and precipitated with 100% EtOH, mussel glycogen (Invitrogen) and 7.5 M NH4OAc. We recovered the precipitate by centrifugation at 20,200g for 15 min at 4 °C. We added a single adenine base to the DNA using Klenow exo– (3′ and 5′ exo minus; Illumina), phenol–chloroform–isoamyl alcohol extracted, and precipitated as above.. The DNA pellet was washed twice with 70% EtOH and resupended in 10 ml of LoTE buffer. Adapters (Illumina) were ligated to ends of the single adenine–tailed DNA following the manufacturer's instructions with the exception that we diluted the adaptor oligonucleotide mix (Illumina) 1/10 before use. We recovered the DNA using a QIAquick PCR Purification Kit (Qiagen) and we enriched the adaptor-modified DNA fragments by PCR using Phusion polymerase (Finnzymes) and PCR primer 1.1 and 2.1 (Illumina) following the manufacturer's instructions (separate 10- and 15-cycle reactions were run). After cycling, we purified the reactions using a QIAquick MiniElute kit (Qiagen) according to the manufacturer's instructions, with 15 μl of elution buffer heated to 50 °C. We quantified the DNA quality with an Agilent DNA 1000 series II assay and a Nanodrop 7500 spectrophotometer using a 1.5-μl aliquot that was diluted to 10 nM.
GEO Accession
GSM3262717