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Sample GSM821396 Query DataSets for GSM821396
Status Public on Mar 27, 2012
Title mES_Dnmt-TKO_H3K27m3_ChIP
Sample type SRA
Source name Embryonic stem cells
Organism Mus musculus
Characteristics origin: Embryonic stem cells
technique: ChIP-sequencing
antibody: H3K27me3
antibody vendor: Millipore
antibody catalog #: 07-449
antibody lot #: DAM-1588246
Growth protocol HCT116 cells were cultured in McCoy's 5A medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (all Gibco/Invitrogen) at 37 °C in 5% CO2 atmosphere.
ES cells were cultured in normal cell culture containing ES-grade serum supplemented with LIF. These cells were grown on gelatin-coated plates
Extracted molecule genomic DNA
Extraction protocol ES cells were crosslinked with 1% formaldehyde for 30 min at room temperature, quenched with 0.125 M glycine and washed at 4C with three buffers: (i) PBS, (ii) buffer of composition 0.25% Triton X-100, 10 mM EDTA, 0.5 mM EGTA, 20 mM HEPES pH 7.6 and (iii) 0.15 M NaCl in HEG buffer (1 mM EDTA, 0.5 mM EGTA, 20 mM HEPES pH 7.6). Cells were then suspended in ChIP incubation buffer (0.15% SDS, 1% Triton X-100, 150 mM NaCl, HEG) and sheared using a Biouptor (Diagenode). Sonicated chromatin was centrifuged for 5 min and then incubated overnight with 2 ug of antibody and protein A/G beads (Santa Cruz). Beads were washed six times with different buffers at 4C: twice with solution of composition 0.1% SDS, 0.1% DOC, 1% Triton, 150 mM NaCl, HEG, once with the solution same as before but with 500 mM NaCl, once with solution of composition 0.25 M LiCl, 0.5% DOC, 0.5% NP-40, HEG and twice with HEG. Precipitated chromatin was eluted with 400 µl of elution buffer (1% SDS, 0.1 M NaHCO3), incubated at 65C for 4 h in the presence of 200 mM NaCl, phenol extracted and precipitated with 20 ug of glycogen at -20C overnight.
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 2000
Description 1 to 30 ng was used for library preparation using the ChIP-seq DNA sample Prep Kit (Illumina, cat# IP-102–1001) using the recommended protocol. After ligation of adapters, a 300 bp band is excised using the E-Gel system (Invitrogen), which correspond to a 234 bp fragment of genomic DNA. PCR enrichment of adapter-ligated DNA was done using 14 cycles of PCR.
Data processing Cluster generation and sequencing-by-synthesis (36 bp) was performed using the Illumina HiSeq2000 platform according to standard protocols of the manufacturer (Illumina). The image files generated by the Genome Analyzer were processed to extract DNA sequence data. Sequences were aligned to the mouse (mm9) reference genome using the Illumina Analysis Pipeline allowing, one mismatch. Only the tags uniquely aligning to the genome were considered for further analysis. Further analysis was performed using the 36 bp sequence reads. The output data were converted to Browser Extensible Data (BED) files for downstream analysis and Wiggle (WIG) files for viewing the data in the UCSC Genome Browser. All sequence analyses were conducted based on the Mus Musculus mm9 genome assembly.
Submission date Oct 24, 2011
Last update date May 15, 2019
Contact name Arjen Brinkman
Organization name Radboud University, Nijmegen Center for Molecular Life Sciences
Department Molecular Biology
Lab Stunnenberg
Street address NCMLS #274, Geert Grooteplein Zuid 30
City Nijmegen
ZIP/Postal code 6525 GA
Country Netherlands
Platform ID GPL13112
Series (1)
GSE28254 Sequential ChIP-bisulfite sequencing enables direct genome-scale investigation of chromatin and DNA methylation cross-talk
SRA SRX105267
BioSample SAMN00752490

Supplementary file Size Download File type/resource
GSM821396_mES_Dnmt-TKO_H3K27m3_ChIP.reads.bed.gz 540.3 Mb (ftp)(http) BED
GSM821396_mES_Dnmt-TKO_H3K27m3_ChIP.reads.wig.gz 64.3 Mb (ftp)(http) WIG
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Raw data are available in SRA
Processed data provided as supplementary file

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