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| Status |
Public on Mar 01, 2013 |
| Title |
CDK8-ChIP enriched DNA, normoxia |
| Sample type |
SRA |
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| Source name |
colorectal cancer cells, normoxia, CDK8 ChIP
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HCT116
treatment: normoxia
chip antibody: CDK8
antibody vendor: Santa Cruz
antibody catalog #: sc-1521
antibody lot#: K1510
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| Treatment protocol |
HCT116 cells were subjected to normoxia or hypoxia for 24 h.
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| Growth protocol |
HCT116 cells were plated in McCoy's 5A Medium supplemented with 10% fetal calf serum 24hrs prior to treatment as indicated.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin immunoprecipitation: Media was removed and the cells were cross-linked by the addition of 1% formaldehyde in PBS for 15 min, followed by the addition of glycine to 0.125 M to quench the formaldehyde. Plates were then washed twice with cold PBS to remove all traces of formaldehyde. Cross-linked cells were harvested by scraping directly into RIPA buffer (150 mM NaCl, 50 mM Tris pH 8, 5 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, protease/phosphatase/HDAC inhibitors) and snap-frozen. Thawed whole-cell lysates were then fragmented by sonication to give DNA fragments of ~200 bp and centrifuged at 12,000 x g to remove insoluble material. The supernatant was quantified using a BCA protein assay kit (Pierce) and diluted to 1 mg/ml with RIPA buffer. For immunoprecipitation, 1 mg of ChIP extract was first pre-cleared by incubation with RIPA-washed Protein-G sepharose beads (GE Healthcare) at 4°C for 2 hr. Pre-cleared supernatants were then mixed with Protein-G sepharose beads that had been blocked for 2 hr with 1 mg/ml bovine serum albumin (Sigma) and immunoprecipitated by incubating overnight at 4°C with the desired antibody. Each ChIP was then washed (5 min each wash) twice with RIPA buffer, four times with IP wash buffer (100 mM Tris pH 8.5, 0.5 M LiCl, 1% NP-40, 1% sodium deoxycholate), twice more with RIPA buffer, twice with TE (10 mM Tris pH 8, 1 mM EDTA), and finally resuspended in 100 μl TE. Immunocomplexes were then eluted by the addition of two volumes of elution buffer (70 mM Tris pH 8, 1 mM EDTA, 1.5% SDS) and incubated at 65°C for 10 min. To reverse cross-linking, NaCl was added to a final concentration of 200 mM, followed by incubation at 65°C for 5 hr. To digest remaining protein, 20 μg proteinase K was added and incubated at 45°C for 30 min. Immunoprecipitated DNA was then purified by phenol/chloroform extraction, ethanol precipitated, and finally dissolved in 0.1x TE. Input DNA was prepared essentially in the same manner, omitting the immunoprecipitation steps.
ChIP-seq library preparation and Illumina sequencing: Twenty nanograms of ChIP-enriched DNA or input DNA (prepared as above) were used for generation of Illumina sequencing libraries using a modified version of the Illumina ChIP-seq protocol. Briefly, DNA fragments were end-repaired using the End-It DNA End-Repair Kit (Epicentre) and then a single "A" base was added using Klenow fragment (New England Biolabs). The fragments were ligated to Illumina Indexed adaptors (TruSeq DNA Sample Prep Kit) using T4 DNA ligase (New England Biolabs). The ligated products were size-selected for 350-450 bp fragments on a 2% agarose gel to remove the unligated adaptors, and were subjected to 18 PCR cycles (Illumina TruSeq primers). PCR product was purified on a 2% agarose gel to retain fragments between 350-450bp. Libraries were quantified using a Qubit fluorometer (Invitrogen) and by quantitative PCR (Kapa Biosystems).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
ChIP-enriched DNA from HCT116 cells under normoxia.
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| Data processing |
Base-calling: Illumina CASAVA 1.8.2.
Filtering: adapter sequences (~13%) were filtered out using the FASTX toolkit 0.0.13.1.
Alignment to GRCh37/hg19: Bowtie 0.12.7 with zero-mismatches and discarding non-unique alignments.
Peak-calling: enriched CDK8 ChIP peak regions were determined using MACS 2.0.9 with both ChIP and control (input) samples, p<0.05, keeping at most two duplicate reads, with an estimated shift-size of 225bp.
ChIP-seq data analysis and visualization: Raw 50-nucleotide ChIP-seq reads were subjected to quality assessment (97% bases ≥ Q30) using FASTQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and adapter sequences (~13%) were filtered out using the FASTX toolkit (http://hannonlab.cshl.edu/fastx_toolkit/index.html). Reads were then aligned to the reference human genome (GRCh37/hg19) using Bowtie 0.12.7 with zero-mismatches and discarding non-unique alignments (~80% uniquely aligned). Enriched CDK8 ChIP peak regions were determined using MACS 2.0.9 (Zhang et al., 2008) with both ChIP and control (input) samples, p<0.05, keeping at most two duplicate reads, with an estimated shift-size of 225bp.
Genome_build: GRCh37/hg19
Supplementary_files_format_and_content: BED files (listing peaks) and bedgraph files were generated by MACS 2.0.9.
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| Submission date |
May 25, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Matthew D Galbraith |
| Organization name |
University of Colorado Anschutz Medical Campus
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| Department |
Pharmacology & Linda Crnic Institute for Down Syndrome
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| Street address |
RC1-N, Mail Stop 8303, Rm. P18-6114 12800 E. 19th Ave.
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| City |
Aurora |
| State/province |
CO |
| ZIP/Postal code |
80045 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE38258 |
CDK8 ChIP-seq from HCT116 cells in normoxia and hypoxia |
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| Relations |
| SRA |
SRX149659 |
| BioSample |
SAMN00998564 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM937561_CDK8_normox_merge_peaks.bed.gz |
269.2 Kb |
(ftp)(http) |
BED |
| GSM937561_CDK8_normox_merge_treat_pileup.bedgraph.gz |
1.2 Gb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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