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| Status |
Public on Dec 18, 2018 |
| Title |
HKC_day7_H3K4me3 ChIP-Seq |
| Sample type |
SRA |
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| Source name |
keratinocytes
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| Organism |
Homo sapiens |
| Characteristics |
cell line: Dombi23
Stage: day7 (late differentiation)
antibody: Diagenode #C15410003
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| Growth protocol |
primary keratinocytes were cultured in Keratinocyte Basal Medium (KBM, Lonza #CC-4131) supplemented with 100 U/mL Penicillin/Streptomycin (Gibco Life Technology #15140122), 0.1 mM ethanolamine (Sigma Aldrich #141-43-5), 0.1 mM O-phosphoethanolamine (Sigma Aldrich #1071-23-4), 0.4% (vol/vol) bovine pituitary extract, 0.5 μg/mL hydrocortisone, 5 μg/mL insulin and 10 ng/mL epidermal growth factor (Lonza #CC-4131). Medium was refreshed every other day. When cells were more than 90% confluent (day 0), differentiation was induced by depletion of growth factors in addition to cell contact inhibition. Cells were collected at four differentiation stages, proliferation (day 0), early differentiation (day 2), mid differentiation (day 4), and late differentiation (day 7) for subsequent experiment. No mycoplasma contamination is found during cell culture.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin was incubated overnight at 4 °C in 1× incubation buffer (0.15% SDS, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, and 20 mM HEPES) supplemented with protease inhibitors and 0.1% BSA. A 50:50 mix of Protein A and G Dynabeads (Invitrogen) were added. Antibodies against H3K4me3 (Diagenode, C15410003, 1 μg), H3K27me3 (Diagenode, C15410069, 1.5 μg) and RUNX1 (ab23980, abcam, 4 μg) were used in each ChIP assay. The beads were washed twice with wash buffer 1 (0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, and 20 mM HEPES), once with wash buffer 2 (wash buffer 1 with 500 mM NaCl), once with wash buffer 3 (250 mM LiCl, 0.5% sodium deoxycholate, 0.5% NP-50, 1 mM EDTA, 0.5 mM EGTA, and 20 mM HEPES), and twice with wash buffer 4 (1 mM EDTA, 0.5 mM EGTA, and 20 mM HEPES). After the washing steps, beads were rotated for 20 min at room temperature in elution buffer (1% SDS and 0.1 M NaHCO3). The supernatant was de-cross-linked with 200 mM NaCl and 100 μg/mL proteinase K for 4 h at 65 °C. De-cross-linked DNA was purified with MinElute PCR Purification columns (Qiagen). DNA amounts were determined with Qubit fluorometric quantification (ThermoFisher Scientific).
The pulled down DNA fragments were proceeded on with library construction using KAPA Hyper Prep Kit (Kapa Biosystems #KK8504) according to the standard protocol. The prepared libraries were then sequenced using the NextSeq 500 (Illumina) according to standard Illumina protocols.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to the self-generated hg19 reference genome using STAR 2.5.0. The code to generate the reference genome: STAR --runThreadN 8 --runMode genomeGenerate --genomeDir /home/self_generated_genome/ --genomeFastaFiles /home/hg19.fa --sjdbGTFfile /home/Homo_sapiens.GRCh37.75_new.gtf --sjdbOverhang 100 The code for mapping: STAR --runThreadN 16 --quantMode GeneCounts --outSAMstrandField intronMotif --outFilterIntronMotifs RemoveNoncanonical --outFilterType BySJout --outSAMtype BAM SortedByCoordinate --outWigType wiggle --outWigStrand Unstranded --genomeDir /home/self_generated_genome --readFilesCommand zcat --readFilesIn $r1file $r2file --outFileNamePrefix /home/mapping
wigToBigWig from UCSC geome browser is used to generate the bigwig files from the wiggle files
peaks.bed files are generated with MACS2 and contain all the peak information.
Genome_build: hg19, Homo_sapiens.GRCh37.75_new.gtf
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| Submission date |
May 02, 2017 |
| 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 |
GPL18573 |
| Series (1) |
| GSE98483 |
p63 controls the enhancer landscape during keratinocyte differentiation |
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| Relations |
| BioSample |
SAMN06883682 |
| SRA |
SRX2779397 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2597291_lane1_H3K4me3-ChIP-Dombi23-day7_peaks.bed.gz |
573.1 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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