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| Status |
Public on Dec 18, 2018 |
| Title |
HKC_day0-1_RNA-Seq |
| Sample type |
SRA |
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| Source name |
keratinocytes
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| Organism |
Homo sapiens |
| Characteristics |
cell line: PKC19
Stage: day0 (proliferation)
antibody: none
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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 |
total RNA |
| Extraction protocol |
Total RNA was isolated using the NucleoSpin RNA kit (MACHEREY-NAGEL #740955.250).
RNA-Seq experiment was performed with the starting material of 500 ng total RNA, to obtain double-strand cDNA (ds-cDNA). After purification with the MinElute Reaction Cleanup Kit (Qiagen #28206), 3 ng ds-cDNA was processed for library construction using KAPA Hyper Prep Kit (Kapa Biosystems #KK8504) according to the standard protocol except that a 15-minute USER enzyme (BioLab # M5505L) incubation step was added before library amplification. The prepared libraries were quantified with the KAPA Library Quantification Kit (Kapa Biosystems #KK4844), and then sequenced in a paired-ended manner using the NextSeq 500 (Illumina) according to standard Illumina protocols.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| 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
ReadsPerGene.out.tab file is generated with STAR 2.5.0 with the option of --quantMode GeneCounts
ReadsPerGene.out.tab file is generated with STAR 2.5.0 and contains the information of read counts per gene.
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 |
SAMN06883693 |
| SRA |
SRX2779386 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2597280_lane1_RNASeq-PKC19-1-day0_ReadsPerGene.out.tab.gz |
312.5 Kb |
(ftp)(http) |
TAB |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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