GSM1479709: PolII ZT 2 WT; Mus musculus; ChIP-Seq
ChIP-seq PolII protocol: Perfused livers were processed for chromatin preparation as described in Ripperger JA, Schibler U (2006). The chromatin samples from the five mice were then pooled, frozen in liquid nitrogen, and stored at -80°C. For the ChIP experiments, the following antibodies were used: anti-RPB2 (Santa Cruz Biotechnology, sc-673-18), anti-H3K4me3 (Abcam, ab8580), and anti-H3K36me3 (Abcam, ab9050). To determine the optimal amounts of each antibody, we performed pilot ChIP assays and determined the enrichment for a set of promoters by real-time qPCR according to Ripperger JA, Schibler U (2006). A total of 1 ml of each chromatin suspension (containing about 60 µg of DNA) was incubated with 10 µg of anti-RPB2, 1.5 µg of anti-H3K36me3, or 1.5 µg of anti-H3K4me3 in buffer A (20 mM Tris/HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA) overnight at 4°C on a rotating wheel. Ten µl of protein A bead suspension (25% slurry in buffer A), pre-blocked with 10 µg/ml of salmon sperm DNA and BSA at 4°C overnight, was then added and the incubation was continued for 1 h at room temperature on a rotating wheel. The beads were then washed with dialysis buffer and ChIP wash buffer as described O'Geen H, Nicolet CM, Blahnik K, Green R, Farnham PJ (2006). Protein-DNA complexes were eluted from the beads, de-cross-linked, and treated with RNase A, and subsequently, with proteinase K, as described O'Geen H, Nicolet CM, Blahnik K, Green R, Farnham PJ (2006). The DNA concentration was determined by fluorometry on the Qubit system (Invitrogen). A total of 10-12 ng DNA were used for the preparation of the library. Libraries for ultra-high-throughput sequencing were prepared with the ChIP-Seq DNA sample kit (Illumina) as recommended by the manufacturer. Libraries for ultra-high-throughput sequencing were prepared with the ChIP-Seq DNA sample kit (Illumina) as recommended by the manufacturer.
GEO Accession
GSM1479709