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| Status |
Public on Nov 17, 2010 |
| Title |
GATA6 ChIP-seq in proliferating cells |
| Sample type |
SRA |
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| Source name |
proliferating Caco-2 cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: human intestinal cell line
cell line: Caco-2
chip antibody: GATA6 (Santa Cruz 7244 and Santa Cruz 9055)
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| Treatment protocol |
For transcription factor ChIP, cells were fixed in 1% formaldehyde for 10 minutes at 37 degrees, then washed with cold PBS and scraped into PBS, and resuspended in ChIP lysis buffer and immunoprecipitated and processed as described by Carroll, et al, 2005, PMID: 16009131 and as described in our current work. Antibodies used were CDX2 (Bethyl BL3194), GATA6 c-20 (Santa Cruz 7244), GATA6 h-92 (Santa Cruz 9055), HNF4α c-19 (Santa Cruz 6556) or HNF4α h-171 (Santa Cruz 8987). For H3K4me2 ChIP, cells were collected in a digestion buffer (50 mM Tris-HCl, pH7.6, 1 mM CaCl2, 0.2% Triton X-100, 5 mM Na butyrate, complete protease inhibitors) and treated with 0.2 U micrococcal nuclease (MNase, Sigma Aldrich) for 8 min at 37°C. The reaction was terminated by adding 5 mM EDTA in 10 mM Tris, pH7.6, and samples were dialyzed in chromatin RIPA buffer (10 mM Tris, pH7.6, 1 mM EDTA, 0.1% SDS, 0.1% Na deoxycholate, 1% Triton X-100) before overnight IP at 4oC with Histone H3K27Ac (Abcam ab4729) or H3K4Me2 (Millipore 07-030) antibodies. IP material was subsequently handled as described below for conventional ChIP of sonicated genomic DNA.
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| Growth protocol |
for ChIP-seq experiments, Caco-2 cells were grown in 10% FBS DMEM and passaged frequently for subconfluent cells or fed every second day for post-confluent "differentiated" cells. Cells were harvested at 50% confluence (proliferating) or 26 days post-confluent (differentiated)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Libraries were constructed using 10 ng each of ChIP and input DNA from up to 3 pooled experiments were processed for deep sequencing according to manufacturer’s instructions (Illumina). Prior to sequencing, qPCR was used to verify that positive and negative control ChIP regions amplified in the linear range.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
chromatin IP against GATA6 in proliferating cells
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| Data processing |
ChIP-Seq: ELAND mapping (hg18) + MACS for CDX2, HNF4, and GATA6 ChIP-Seq data. ELAND mapping (hg18) + NPS for H3K4me2 Mnase ChIP-Seq data.
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| Submission date |
Aug 05, 2010 |
| Last update date |
May 15, 2019 |
| Contact name |
Michael P Verzi |
| Organization name |
Rutgers, the State University of New Jersey
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| Department |
Genetics
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| Lab |
Verzi
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| Street address |
145 Bevier Road
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| City |
Piscataway |
| State/province |
NJ |
| ZIP/Postal code |
08854 |
| Country |
USA |
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| Platform ID |
GPL9115 |
| Series (1) |
| GSE23436 |
Histone methylation and transcription factor binding during intestinal cell differentation |
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| Relations |
| SRA |
SRX026265 |
| BioSample |
SAMN00109936 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM575226_GATA6_cycling.bed.gz |
49.4 Mb |
(ftp)(http) |
BED |
| GSM575226_GATA6_cycling_pval1e-2_peaks.txt.gz |
2.7 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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