Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
ESR1

Cell type

Cell type Class
Breast
Cell type
MDA-MB-231
Primary Tissue
Breast
Site of Extraction
Effusion, Pleural
Tissue Diagnosis
Adenocarcinoma

Attributes by original data submitter

Sample

source_name
ERα_ChIP-seq_231ERαLQ_0min
cell line background
MDA-MB-231
transduced with
ERα L540Q
treated with
E2, 100 nM, 45 min

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were grown to ~80% confluence and were crosslinked with 1% formaldehyde in PBS at 37°C for 10 min. The crosslinking reaction was quenched by adding glycine to a final concentration of 125 mM. The cells were collected by scraping in 1x PBS containing 1x complete protease inhibitor cocktail (Roche, 11697498001). The cells were pelleted by brief centrifugation in a microcentrifuge and lysed by pipetting in Farnham Lysis Buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40, 1 mM DTT, 1x complete protease inhibitor cocktail). The nuclei were collected by brief centrifugation in a microcentrifuge and resuspended in SDS Lysis Buffer (Tris•HCl pH 7.9, 1% SDS, 10 mM EDTA, 50 mM, 1 mM DTT, 1x complete protease inhibitor cocktail) by pipetting and incubation on ice for 10 min. The chromatin was then sheared to ~200-500 bp DNA fragments by sonication using a Bioruptor Plus sonicator (Diagenode) for 25-30 cycles of 30 seconds on and 30 seconds off. The solubilized sonicated chromatin was precleared with Protein A or Protein G Dynabeads (Invitrogen, 10001D and 10003D, respectively) and incubated overnight with 8 μL of polyclonal antiserum or 2.5 to 5.0 μg of commercial antibody. The immune complexes from the ChIP were precipitated by the addition of Protein A or Protein G Dynabeads (depending on the antibody used) and washed once with each of the following wash buffer in sequence: (1) Low Salt Wash Buffer (20 mM Tris•HCl pH 7.9, 2 mM EDTA, 125 mM NaCl, 0.05% SDS, 1% Triton X-100, 1x complete protease inhibitor cocktail); (2) High Salt Wash Buffer (20 mM Tris•HCl pH 7.9, 2 mM EDTA, 500 mM NaCl, 0.05% SDS, 1% Triton X-100, 1x complete protease inhibitor cocktail); (3) LiCl Wash Buffer (10 mM Tris•HCl pH 7.9, 1 mM EDTA, 250 mM LiCl, 1% NP-40, 1% sodium deoxycholate, 1x complete protease inhibitor cocktail); and (4) 1x Tris-EDTA (TE) containing 1x complete protease inhibitor cocktail. The precipitated immune complexes were transferred to a new tube in 1x TE/complete protease inhibitor cocktail before elution of the genomic DNA fragments in Elution Buffer [40 mM Tris•HCl pH 7.9, 10 mM EDTA, 100 mM NaCl, 100 mM NaHCO3, 1% SDS, and 50 μg Proteinase K (Life Technologies, 2542)] for 2 hours at 55°C. The crosslinks were reversed by incubating overnight at 65 °C, and the genomic DNA was purified using phenol:chloroform:isoamyl acid extraction (Sigma, P2069) followed by ethanol precipitation. Input DNA or ChIPed DNA was subjected to additional purification using Agencourt AMPure XP beads (Beckman Coulter, A63881). Two and a half to 10 ng of purified genomic DNA was subjected to end repair using an end-repair mix (Enzymatics, Y9140-LC-L) and 0.1 mM dNTPs. A single dA base was added to the end repaired DNA using Klenow 35 Exo-minus (Enzymatics, P7010-HC-L), 1x Blue Buffer (Enzymatics, B0110), and 0.2 mM dATP to facilitate adaptor ligation. TruSeq DNA Sample Prep Kit adaptors (custom synthesized by IDT, HPLC purified) were partially annealed to form double-stranded adaptors on one terminus, and annealed to insert DNA through double strand DNA-DNA ligation using T4 DNA ligase (Enzymatics, L6030-HC-L) in DNA Rapid Ligase Buffer (Enzymatics, B1010). The adaptor-ligated DNA was amplified by PCR for 6 - 11 cycles and purified by electrophoresis on a 1% agarose gel containing SYBR Gold (Invitrogen, S11494). The DNA was excised from the agarose gel and eluted using a QIAquick Gel Extraction Kit (Qiagen, 28706). The quality of the library was assessed using a D1000 ScreenTape (Agilent, 5067-5582) on a 2200 TapeStation (Agilent) and quantified using a Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Q32851). The libraries with unique adaptor barcodes were multiplexed and sequenced on an Illumina HiSeq 2000 (single-end, 50 base reads).

Sequencing Platform

instrument_model
Illumina HiSeq 2000

hg19

Number of total reads
16294639
Reads aligned (%)
85.3
Duplicates removed (%)
8.9
Number of peaks
10126 (qval < 1E-05)

hg38

Number of total reads
16294639
Reads aligned (%)
86.7
Duplicates removed (%)
8.1
Number of peaks
9796 (qval < 1E-05)

Base call quality data from DBCLS SRA