Antigen

Antigen Class

RNA polymerase

Antigen

RNA polymerase II

Cell type

Cell type Class

Liver

Cell type

Liver

MeSH Description

A large lobed glandular organ in the abdomen of vertebrates that is responsible for detoxification, metabolism, synthesis and storage of various substances.

Sample

source_name

Liver, WT, ZT 26, PolII ChIP

strain/background

C57/BL6

genotype/variation

WT

gender

male

feeding

night-restricted feeding

tissue

liver

time point

ZT 26

technique

PolII ChIP-seq

chip antibody

anti-PolII

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

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.

Sequencing Platform

instrument_model

Illumina HiSeq 2500

mm10

Number of total reads

191096558

Reads aligned (%)

92.0

Duplicates removed (%)

49.0

Number of peaks

37011 (qval < 1E-05)

mm9

Number of total reads

191096558

Reads aligned (%)

91.9

Duplicates removed (%)

49.0

Number of peaks

37225 (qval < 1E-05)