GSM1382475: Input DNA, EtOH, rep2, MCF7; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Input control
Antigen
Input control
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_Input DNA_EtOH
cell line
MCF7
cell type
human breast adenocarcinoma
treated with
vehicle (EtOH) for 30 min
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
MCF-7 cells were cross-linked 10 min at room temperature (1% formaldehyde in 1X PBS pH 7.4). ChIP assays were performed as described previously (Svotelis A et al., 2009), except for the chromatin preparation. Briefly, permeabilized cells were incubated 2 min at 37oC in digestion buffer (50 mM Tris-HCl pH 8.0, 1 mM CaCl2, 0.2% Triton and protease inhibitors), before the addition of Micrococcal Nuclease (MNase, USB Corporation) 5 min at 37oC. Then the digestion reactions were stopped (5 mM EDTA, 10 mM Tris-HCl pH 8 and 0.5% SDS and protease inhibitors) and chromatin sample were briefly sonicated to fragment insoluble chromatin from the pellet without affecting the fragmentation of mononucleosomal fragments. No visible pellet remained after a centrifugation at 15,000 g at 4oC for 10 min. The digestion and the full extraction of chromatin were verified on gel electrophoresis prior to the immunoprecipitation of the chromatin. The sequencing libraries were performed according to the Illumina library preparation protocol except for the size-selection of DNA fragments and for DNA recovery that were executed as described previously (Rodrigue S et al., 2010). Quant-iT PicroGreen dsDNA Assay Kit (Invitrogen) was used to quantify ChIP-DNA and 10 ng was used for the preparation of the sequencing libraries. The size-selection of nucleosome fragments, to exclude subnucleosomal and polynucleosomal particules, was executed in parallel with the preparation of libraries and was performed using solid-phase paramagnetic beads technology (AMPure XP PCR Purification systems, Beckman Coulter), since it allows a more precise size-selection of the DNA fragments than conventional gel electrophoresis and minimizes loss of sample, which is particularly relevant for the dilute samples frequently obtained after ChIP experiments. It was thus used for all the DNA-recovery steps of the libraries preparation as well as to eliminate potential primer dimers or primers and linkers in excess (shorter than the libraries of nucleosome fragments) after the final amplification of the libraries. Importantly, this final step was monitored by qPCR and stopped in the exponential-phase to avoid PCR-duplicates. The quantification of the final libraries, the absence of primer-dimers as well as the validity of the size-selection were determined using the 2100 Bioanalyzer instrument (Agilent Technologies). To ensure reproducibility, two biological replicates of every mark or H2A.Z or control input were single-end sequenced with 40 nucleotides reads using an Illumina HiSeq 2000 platform by the MIT MicroBioCenter.