GSM1544113: HMEC-hTERT Input; 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
HMEC
Primary Tissue
Breast
Tissue Diagnosis
Normal
Attributes by original data submitter
Sample
source_name
cell line
cell line
HMEC-hTERT
protocol
sonication
chip antibody
none (input)
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Genomic DNA was sheared to a mean fragment length of 300-500 bp through sonication. Methylated-DNA precipitation (MeDP) was carried out using the MethylMiner Methylated DNA Enrichment Kit (Life Technologies) from 1µg of sheared DNA. Pools of 5 independent experiments, library preparation and high throughput sequencing (single-end 50 bp sequencing on Illumina HiSeq 2000) were performed at Beijing Genomics Institute (Hong-Kong, China). MBD2 Chromatin Immunoprecipitations (ChIP) were performed as previously described (Auriol et al. Nucleic Acids Research 2005). Briefly, sheared chromatin (with a mean fragment length between 300 and 500 bp) was obtained by sonication of formaldehyde cross-linked nuclei. ChIP was then performed with a custom-made rabbit polyclonal serum obtained after immunization with peptides corresponding to the N-terminal part of the MBD2 protein (Covalab, Villeurbanne, Lyon) (Perriaud et al. Curr. Pharm. Des 2013) using the ChIP Assay Kit (Merck Millipore, Saint-Quentin-en-Yvelines, France) as specified by the manufacturer’s instruction. Precipitated DNA was finally purified using NucleoSpin Gel and PCR Clean-up kit, (Macherey-Nagel) according to the manufacturer’s protocol “DNA clean-up of samples containing SDS”. DNA-end was repaired to overhang a 3'-dA, then adapters were ligated to the end DNA fragments. DNA fragments with proper size (usually 100-300bp, including adaptor sequence) were selected after PCR amplification.