Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Input control
Antigen
Input control

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.

Attributes by original data submitter

Sample

source_name
Liver, Bmal1 KO, ZT 22, input
strain/background
C57/BL6
genotype/variation
Bmal1 KO
gender
male
feeding
night-restricted feeding
tissue
liver
time point
ZT 22
chip antibody
none

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
DNase1-seq protocol: Mouse liver nuclei were prepared as described in (Tian and Schibler 1991). Freshly prepared nuclei were suspended in ice-cold Ψ-buffer (11 mM KPO4 pH 7.4, 108 mM KCl, 22 mM NaCl, 5mM MgCl, 1 mM CaCl2, 1 mM DTT) and pelleted. 5x10^6 nuclei were suspended in 200 μl of Ψ-buffer supplemented with 0.2% of NP40 and 1 u/ml of DNaseI (DPFF Worthington Biochemical Corporation). DNase1 digestion was performed for 6 minutes in room temperature and the reaction was stopped by adding 200 μl of lysis buffer (50mM Tris-HCl pH 8, 20 mM EDTA, 1% SDS, 200 μg/ml proteinaseK). Protease digestion was performed overnight at 55 oC. RNaseA (100 μg/ml) was then added and samples were incubated at 37 oC for an hour. DNA was then extracted twice with phenol-chloroform and precipitated with isopropanol in the presence of 0.5 M NaCl. DNAs were dissolved in 5 mM Tris-HCl pH 8.DNAs from 4 animals were pooled and 75 μg of DNA was loaded on 11 ml 10%-50% sucrose gradient in STE buffer (1M NaCl, 20 mM Tris-HCl pH 8, 5 mM EDTA) and centrifuged at 30000 rpm for 16 hours at 20 oC (SW 40 Ti rotor, Beckman Coulter Inc). The sucrose gradients were then fractionated and DNA was precipitated by two volumes of ethanol in the presence of 5 μg of glycogen. Fractions containing DNA sized around 300bp were pooled and used for Illumina library preparation. 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 antibody was used: anti-RPB2 (Santa Cruz Biotechnology, sc-673-18). To determine the optimal amounts of 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 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 in 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 in 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. ChIP-seq H3K27ac protocol: ChIPs were performed according to the method described by Reddy et al. (2009) with a few modifications. The 100 uL chromatin aliquots were used for each IP and diluted with 900 ul of RIPA buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS in PBS at pH 7.4) and added to dynal magnetic beads conjugated with (sheep anti-mouse IgG Dynabeads, Invitrogen, Cat no: 110-31) pre-treated with 3 ul of polyclonal antibody for H3K27ac (Active Motif, Cat no: 39135) for immunoprecipitation of specific complexes. The samples were incubated overnight at 4°C on rotator, then magnetic beads washed 7 times with lithiumchloride wash buffer (100mM Tris at pH 7.5, 500mM LiCl, 1% NP-40 and 1% sodiumdeoxycholate) and finally once with 1X TE buffer (10mM Tris-HCl at pH 7.5, 0.1mM Na2EDTA). The chromatin complex was eluted using elution buffer (1% SDS, 0.1M NaHCO3) for 1 h at 65°C using eppendorf thermo-mixer. The chromatin was then de-crosslinked overnight at 65°C and ChIP DNA purified using Qiagen PCR purification kit and eluted in 50 ul of elution buffer. For qPCR reaction, 1.5 ul of 1/10 diluted ChIP DNA is used. 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
236000342
Reads aligned (%)
92.9
Duplicates removed (%)
26.7
Number of peaks
609 (qval < 1E-05)

mm9

Number of total reads
236000342
Reads aligned (%)
92.7
Duplicates removed (%)
27.2
Number of peaks
634 (qval < 1E-05)

Base call quality data from DBCLS SRA