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| Status |
Public on May 07, 2013 |
| Title |
C_29_GRHL2_ChIPseq |
| Sample type |
SRA |
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| Source name |
Bronchial Epithelial Cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Bronchial Epithelial Cells
donor: C29
chip antibody: GRHL2
chip antibody manufacturer: Sigma
chip antibody catalog #: HPA004820
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| Growth protocol |
Cells were seeded into plastic dishes coated with bovine collagen (PureCol, Advanced BioMatrix, 5005-B) in bronchial epithelial cell growth medium (BEGM) containing 0.11mM Ca++ and 25 ng/ml EGF. Confluent cultures were passaged to P2 in dishes without collagen, and grown to confluence.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
20 million cells were crosslinked for 10 minutes in 1% formaldehyde followed by 5 minutes of quenching in 0.125 M glycine. Cells were then washed with cold PBS pH7.4, and collected in the presence of cold cell lysis buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40, and Roche Complete Protease Inhibitor Cocktail Cat #11836145001). Nuclei were pelleted by centrifugation at 2,000 rpm for 5 minutes at 4C, and the supertanant discarded. Sheared chromatin was then prepared by sonicating the nuclei in the presence of RIPA buffer (1 x PBS pH 7.4, 1% NP-40, 0.5% NaDOC, Roche Protease Inhibitor Cocktail), followed by centrifugation to remove cell debris. To collected GRHL2-bound DNA, the chromatin was immunoprecipitated with an anti-GRHL2 antibody (Sigma HPA004820), the formaldehyde crosslinks were reversed by heating overnight at 65C, and the DNA was collected using Qiagen PCR cleanup columns. For input control samples, reverse-crosslinked DNA was isolated from an aliquot of the sonicated but not immunoprecipitated chromatin.
DNA ends were blunted using a mixture of Klenow DNA polymerase, T4 DNA polymerase, and T4 polynucleotide kinase (PNK). Three-prime adenine was then added using 3'-5' exo- Klenow fragment, Illumina Y-adapters were ligated using T4 DNA ligase. The ligated DNA was gel size selected for 150-300 bp fragments, and PCR-amplified to make the final sequencing library. Samples were sequenced on an Illumina HiSeq 2000.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
GRHL2 ChIP-seq
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| Data processing |
Base-calling was performed on instrument using CASAVA software
Reads were aligned to the human genome (version hg19) using Bowtie version 0.12.8.
GRHL2 binding sites were called using the MACS peak caller with a bandwidth of 300 bp, an model fold parameter of 15, and a p-value threshold of 1E-10.
Genome_build: hg19
Supplementary_files_format_and_content: bedgraph files of genome-wide sequenced tag density.
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| Submission date |
Apr 18, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Timothy E Reddy |
| E-mail(s) |
tim.reddy@duke.edu
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| Organization name |
Duke University
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| Department |
Department of Biostatistics & Bioinformatics
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| Lab |
ReddyLab
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| Street address |
2347 CIEMAS, 101 Science Drive
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| City |
Durham |
| State/province |
NC |
| ZIP/Postal code |
27708 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE46194 |
Multiple roles for Grainyhead-like transcription factors in the establishment and maintenance of human mucociliary airway epithelium (ChIP-Seq) |
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| Relations |
| BioSample |
SAMN02048836 |
| SRA |
SRX268450 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1125982_SL20749.bedgraph.gz |
24.0 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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