Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
ARNT

Cell type

Cell type Class
Breast
Cell type
MCF-7
Primary Tissue
Breast
Site of Extraction
Pleura
Tissue Diagnosis
Adenocarcinoma

Attributes by original data submitter

Sample

source_name
MCF7_control_HIF1bChIPseq
tissue source
breast
cell line
MCF7
cell type
Breast cancer cell line
treatment
DMSO 72h, 0.5% O2 16h
type of sample
ChIP
chip antibody
ARNT/HIF-1β monoclonal antibody (NB100-124, Novus)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were incubated in hypoxic conditions for 16 hours. Cultured cells were subsequently immediately fixed by adding 1% Formaldehyde (16% Formaldehyde (w/v), Methanol-free, Thermo Scientific) directly in the medium and incubating for 8 minutes. Fixed cells were incubated with 150 µM of glycine for 5 min to revert the cross-links, washed twice with ice-cold PBS 0.5% Triton-X100, scraped and collected by centrifugation (1000 ×G 5min at 4°C). The pellet was resuspended in 1400 µL of RIPA buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 2 mM EDTA pH 8, 1% Triton-X100, 0.5% Sodium deoxycholate, 1% SDS, 1% protease inhibitors) and transferred in a new eppendorf tube. The chromatin was sonicated for 3 min by using a Branson 250 Digital Sonifier with 0.7 s 'On' and 1.3 s 'Off' pulses at 40% power amplitude, yielding a size of 100 to 500 bp. The sample was kept ice-cold at all times during the sonication. The samples were centrifuged (10 min at 16000 ×G at 4°C) and the supernatant were transferred in a new eppendorf tube. The protein concentration was assessed using a BCA assay. Fifty µL of shared chromatin was used as “input” and 1.4 µg of primary ARNT/HIF-1β monoclonal antibody (NB100-124, Novus) per 1 mg of protein was added to the remainder of the chromatin, and incubated overnight at 4°C in a rotator. Pierce Protein A/G Magnetic Beads (Life Technologies) were added to the samples in a volume that is 4X the volume of the primary Ab and incubated at 4°C for at least 5 hours. A/G Magnetic Beads were collected and the samples were washed 5 times with the washing buffer (50 mM Tris-HCl, 200 mM LiCl, 2 mM EDTA, pH 8, 1% Triton, 0.5% Sodium deoxycholate, 0.1% SDS, 1% protease inhibitors), and twice with TE buffer. The A/G magnetic beads were resuspended in 50 µL of TE buffer, and 1.5 µL of RNAse A (200 units, NEB, Ipswich, MA, USA) were added to the A/G beads samples and to the input, incubated for 10 minutes at 37°C. After addition of 1.5 µL of proteinase K (200 units) and overnight incubation at 65°C, the DNA was purified using 1.8´ volume of Agencourt AMPure XP (Beckman Coulter) according to the manufactory instructions Five µg of input and all of the immunoprecipitated DNA were converted into sequencing libraries using the NEBNext Ultra DNA library prep kit.

Sequencing Platform

instrument_model
Illumina HiSeq 2000

hg19

Number of total reads
9350465
Reads aligned (%)
44.0
Duplicates removed (%)
54.5
Number of peaks
1905 (qval < 1E-05)

hg38

Number of total reads
9350465
Reads aligned (%)
45.5
Duplicates removed (%)
53.4
Number of peaks
1888 (qval < 1E-05)

Base call quality data from DBCLS SRA