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| Status |
Public on Oct 26, 2012 |
| Title |
ChIP-seq_HCT116_DKO_H3K27me3 |
| Sample type |
SRA |
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| Source name |
HCT116 with homozygous deletion of DNMT3b and DNMT1 (Rhee et al Nature 2002)
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| Organism |
Homo sapiens |
| Characteristics |
technique: ChIP-sequencing
starting molecule: chromatin
antibody/capture: anti-H3K27me3 (Millipore, 07-449)
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| Treatment protocol |
No specific treatments
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| Growth protocol |
HCT116 WT and DKO 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.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
HCT116 cells were crosslinked with 1% formaldehyde for 30 min at room temperature, quenched with 0.125 M glycine and washed at 4°C 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 3 µg of antibody and protein A/G beads (Santa Cruz). Beads were washed six times with different buffers at 4°C: 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 65°C for 4 h in the presence of 200 mM NaCl, phenol extracted and precipitated with 20 µg of glycogen at -20°C overnight.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Data processing |
Cluster generation and sequencing-by-synthesis (36 bp) was performed using the Illumina Genome Analyzer IIx (GAIIx) platform according to standard protocols of the manufacturer (Illumina). The image files generated by the Genome Analyzer were processed to extract DNA sequence data. Sequence reads were aligned to the human (hg18) reference genome using the Illumina Analysis Pipeline allowing, one mismatch. Only the 36 bp sequence reads uniquely aligning to the genome were considered for further analysis. 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 Homo Sapiens hg18 genome assembly.
Genome_build: hg18
Supplementary_files_format_and_content: The file "MergedPeaks_normalized_tagdensity.gz" contains processed data for all patient samples. It is a tab-delimited text file. MethylCap-seq was performed on 24 micro-dissected tumors and an equal number of matched microscopically normal tissue samples (48 DNA methylation profiles in total). Enriched regions (peaks) were called for each sample on the basis of a Poisson distribution of overlapping sequence reads within a dynamic window. A false discovery rate (FDR) was calculated relative to the total covered sequence, and peaks with an FDR of ≤10-6 were selected. All peaks from the 48 samples were merged. Per sample the number of tags overlapping each region of interest was calculated and normalized by dividing by the total number of aligned reads of the sample, and the length of the peak.
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| Submission date |
Jul 03, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Arjen Brinkman |
| E-mail(s) |
arjen.brinkman@gmail.com
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| Organization name |
Radboud University, Nijmegen Center for Molecular Life Sciences
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| Department |
Molecular Biology
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| Lab |
Stunnenberg
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| Street address |
NCMLS #274, Geert Grooteplein Zuid 30
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| City |
Nijmegen |
| ZIP/Postal code |
6525 GA |
| Country |
Netherlands |
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| Platform ID |
GPL10999 |
| Series (1) |
| GSE39068 |
Comparative genome-wide DNA methylation analysis of colorectal tumor and matched normal tissues |
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| Relations |
| SRA |
SRX157411 |
| BioSample |
SAMN01085280 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM955165_HCT116_DKO_H3K27me3.reads.bed.gz |
61.4 Mb |
(ftp)(http) |
BED |
| GSM955165_HCT116_DKO_H3K27me3.wig.gz |
19.8 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
| Processed data are available on Series record |
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