Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
ESR1

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
breast cancer cell line
cell line
MCF-7
integrated vectors
pcDNA6/TR and pcDNA4/TO -MKL1 deltaN200, Invitrogen
hormone treatment
10-8M Estradiol (Sigma)
chip antibody
HC-20, sc-543, Santa Cruz Biotechnology

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were and lysed for 10 min in ice in 300 µl of lysis buffer [10 mM EDTA, 50 mM Tris-HCl (pH 8.0), 0.1% SDS, 0.5% Empigen BB (Sigma)]. Crosslinked chromatin was sheared to an average size of 400 bp by sonication for 15 min with 30secs on/off cycles at full power in a BioRuptor apparatus (Diagenode). Following 10 minutes of centrifugation at 10,000 x g, 30 µl were kept as input fraction and the remainder diluted 5 fold in IP buffer (2 mM EDTA, 100 mM NaCl, 20 mM Tris-HCl [pH 8.1], and 0.5%Triton X-100) before proceeding to immunoprecipitation overnight at 4°C under rocking using 2 µg of antibodies and 5 µg of yeast tRNAs. Complexes were recovered after 3 hr incubation at 4°C with 5 µg of yeast tRNAs and 100 µl of a 50% protein A or protein G-Sepharose beads slurry (Amersham Pharmacia Biosciences). Precipitates were then serially washed, using 500 µl of Washing Buffers (WB) I [2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 0.1% SDS, 1% Triton X-100, 150 mM NaCl], WB II [2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 0.1% SDS, 1% Triton X-100, 500 mM NaCl], WB III [1 mM EDTA, 10 mM Tris-HCl (pH 8.1), 1% NP-40, 1% Deoxycholate, 0.25 M LiCl] and then twice with 1 mM EDTA, 10 mM Tris-HCl (pH 8.1). Precipitated complexes were removed from the beads through two sequential incubations of 10 min with 50 µl of 1% SDS, 0.1 M NaHCO3. Crosslink was reversed by an overnight incubation at 65°C with 50 µg of Proteinase K (Sigma). Following a subsequent incubation of the samples with 2.5 µg RNAse (Sigma) for 1h at 37°C, the DNA was then purified on NucleoSpin™ columns (Macherey-Nagel) using NTB buffer and eluted in 40 µl of elution buffer. Efficiency of the ChIP assay was evaluated using qPCR positive and negative controls before proceeding to subsequent steps. For each ChIP conditions, from 18 to 20 individually prepared samples were pooled and submitted for library preparation. Libraries were prepared according to Illumina's instructions by IGBMC sequencing facility (Strasbourg, France)

Sequencing Platform

instrument_model
Illumina HiSeq 2000

hg19

Number of total reads
38681817
Reads aligned (%)
44.0
Duplicates removed (%)
54.4
Number of peaks
1187 (qval < 1E-05)

hg38

Number of total reads
38681817
Reads aligned (%)
46.1
Duplicates removed (%)
52.7
Number of peaks
1087 (qval < 1E-05)

Base call quality data from DBCLS SRA