Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
Arntl

Cell type

Cell type Class
Embryonic fibroblast
Cell type
NIH/3T3
Primary Tissue
Embryo
Tissue Diagnosis
Normal

Attributes by original data submitter

Sample

source_name
NIH3T3 cells, 20h, BMAL1 ChIP
cell line
NIH3T3
treatment
dexamethasone synchronized
time post-dex synchronization
20h
chip antibody
anti-BMAL1

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Anti-BMAL1 and anti-CLOCK antibodies were kindly provided by Prof. Michael Brunner (University of Heidelberg). For chromatin preparation, medium of two confluent 15 cm dishes (approximately 10,000,000 cells each) per timepoint was removed, 6 ml PBS+1% fresh formaldehyde were added and cells were placed for 5' at 37°C in the incubator. Cells were washed twice with ice cold PBS and the reaction was stopped with 1.5 ml stop solution (125 mM glycine or 100 mM Tris pH 9.4, 10 mM DTT). Cells were scraped off the dish, transfered to 2 ml tubes, kept on ice for 15', then at 30°C for 15' and vortexed. Lysates were spun down 1' at 2500 rpm, 4°C, supernatant was removed. 500 µl sol II (10 mM EDTA, 1 mM EGTA, 10 mM HEPES pH 6.8, 0.25% Tx-100) were added, tubes were vortexed to resuspend and spun down 5' at 2500 rpm, 4°C. Supernatant was removed, 500 µl solIII (1mM EDTA, 1mM EGTA, 10 mM HEPES pH 6.8, 200 mM NaCl) were added. Tubes were vortexed to resuspend, spun down 5' at 2500 rpm, 4°C and supernatant was removed. The nuclei from the two plates per sample were united in 300 µl nuclei resuspension buffer (22 mM Tris pH 7.4, 165 mM NaCl, 2.2 mM EDTA) + 1% SDS and sonicated 20 min (30" on/off cycles) in a Bioruptor ultrasonicator 4°C water bath (Diagenode) to obtain a fragment size of approx. 250 bp. Chromatin was diluted in 2700 µl chromatin dilution buffer (20 mM Tris pH 7.4, 150 mM NaCl, 2 mM EDTA, 1.1% Tx-100) and aliquoted (500 µl per IP + 5 µl Input). To submit DNA purified from ChIP samples for sequencing, we pooled several IPs to collect 10ng of DNA. We precipitated the DNA using standard sodium acetate precipitation to resuspend it in a small volume and measured the amount of DNA on a Qubit 2.0 fluorometer (Life Technologies). Sample DNA as well as input DNA as control were submitted for Illumina deep sequencing at the Lausanne Genomic Facility (University of Lausanne). A total of 5-10 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.

Sequencing Platform

instrument_model
Illumina Genome Analyzer II

mm10

Number of total reads
7803920
Reads aligned (%)
99.5
Duplicates removed (%)
0.1
Number of peaks
409 (qval < 1E-05)

mm9

Number of total reads
7803920
Reads aligned (%)
99.5
Duplicates removed (%)
0.1
Number of peaks
414 (qval < 1E-05)

Base call quality data from DBCLS SRA