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Status |
Public on May 01, 2015 |
Title |
H3K27ac_day0 |
Sample type |
SRA |
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Source name |
Keratinocytes
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Organism |
Homo sapiens |
Characteristics |
chip antibody: H3K27ac differentiation day: 0 cell type: Keratinocytes
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Treatment protocol |
Differentiation of keratinocyte cultures was induced by cell contact inhibition and excluding several growth factor supplements: bovine pituitary extract (Bio Whittaker), EGF (Sigma), insulin (Sigma), and hydrocortisone (Calbiochem) from the medium. The medium was changed every second day, and before harvesting of the RNA and chromatin. Cells were collected on day 0, and on days 2, 4 and 7 after induction of differentiation.
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Growth protocol |
Skin biopsies were taken from the abdomen of healthy volunteers to set up the primary keratinocyte culture (Rheinwald and Green 1977). Keratinocytes were cultured in Keratinocyte Growth Medium (KGM) under undifferentiated conditions as previously described (Rinne et al. 2008).
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Extracted molecule |
genomic DNA |
Extraction protocol |
ChIP-sequencing datasets were generated with an antibody recognizing the alpha isoforms of p63 (H129, Santa Cruz), an antibody that detects all forms of the large subunit of RNA polymerase II (8WG16, Santa Cruz), and a H3K27ac antibody recognizing the acetylation of the lysine 27 residue of Histone H3 (H3K27ac, Diagenode). RNAPII, p63 and H3K27ac ChIP experiments were performed as previously described (Denissov et al. 2007; Kouwenhoven et al. 2010; Smeenk et al. 2008), with a minor change of using magnetic beads (Novex by Life Technologies, Cat. No. 10008D and 10009D). DNA samples were prepared for sequencing by end repair of 6 ng total DNA (p63 ChIP-seq, and RNAPII ChIP-seq samples) and 10 ng total DNA (H3K27ac ChIP-seq samples) as measured by Qubit (Invitrogen). Adaptors were ligated to the DNA fragments, followed by a pre-PCR of 4 cycles, size selection (∼300 bp) and subsequently 11 cycles of PCR amplification.
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Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina HiSeq 2000 |
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Data processing |
42bp reads left after demultiplexing of original 50bp reads were mapped to the human GRCh37 genome (hg19; assembly February 2009) using BWA 0.6.1 with standard parameters. All duplicate reads and reads mapping to multiple locations were removed. All p63, H3K27ac and RNAPII ChIP-seq datasets (BAM files) were normalized to the same sequencing depth by randomly removing aligned reads. P63 peaks were called using MACS2 (2.0.10.20120913) with default settings and a P value threshold of 1E-9 using input genomic DNA as background control. H3K27ac peaks were called using MACS2 (2.0.10.20120913) using default settings for broad peak calling and a P value threshold of 1E-9 using input genomic DNA as background control. RNAPII occupancy in RefSeq gene bodies calculated as Reads Per Kilobase per Million mapped reads (RPKM). Wiggle tracks were generated from normalized BAM files. Genome_build: hg19 Supplementary_files_format_and_content: Gzip-compressed wiggle files that can be uploaded to the UCSC genome browser as custom tracks. Supplementary_files_format_and_content: MACS2 .xls output file with peak data. Supplementary_files_format_and_content: Tab-separated RefSeq RNAPII gene body occupancy matrix containing genomic location, strand, RefSeq transcript ID, RefSeq gene ID (HUGO gene symbol), and the RPKM values for days 0, 2, 4 & 7 respectively.
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Submission date |
Jul 28, 2014 |
Last update date |
Jun 14, 2022 |
Contact name |
Jo Huiqing Zhou |
E-mail(s) |
jo.zhou@radboudumc.nl
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Organization name |
Radboud University
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Street address |
Geert Grooteplein 26/28
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City |
Nijmegen |
ZIP/Postal code |
6525GA |
Country |
Netherlands |
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Platform ID |
GPL11154 |
Series (2) |
GSE59824 |
Transcription factor p63 bookmarks genomic loci in epithelial cells and regulates a subset of target genes during epidermal differentiation through dynamic enhancers (ChIP-Seq) |
GSE59827 |
Transcription factor p63 bookmarks genomic loci in epithelial cells and regulates a subset of target genes during epidermal differentiation through dynamic enhancers |
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Relations |
BioSample |
SAMN02943048 |
SRA |
SRX663241 |
Supplementary file |
Size |
Download |
File type/resource |
GSM1446919_HKC_L1_ChIP_H3K27ac_day0.wig.gz |
82.6 Mb |
(ftp)(http) |
WIG |
GSM1446919_HKC_L1_ChIP_H3K27ac_day0_broadpeaks.txt.gz |
2.7 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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