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| Status |
Public on May 01, 2015 |
| Title |
HKC_L1_day2_RNAseq |
| Sample type |
SRA |
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| Source name |
Keratinocytes
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HKC L1
differentiation day: 2
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 |
total RNA |
| Extraction protocol |
RNA extraction was carried out using the NucleoSpin RNA II kit (740955.50; MACHEREY-NAGEL, Düren, Germany).
cDNA synthesis from 1 μg total RNA as control for RNA-seq was carried out using the iScript cDNA synthesis kit (Bio-Rad; 170-8891, CA, Hercules, USA). Starting amount of RNA-seq sample was 1 μg. rRNA depletion was performed with the Ribo-Zero rRNA Removal Kit (Human/Mouse/Rat; cat. no. MRZH11124, Epicentre, Madison, WI, USA) according to the manufacturer's instructions. RNA fragmentation reactions were performed using fragmentation buffer (5×; 200 mM Tris-Ac, 500 mM Potassium-Ac, 150 mM Magnesium-Ac) in a final concentration of 1× per reaction. Each 50 μl fragmentation reaction was incubated at 95°C for 1.5 minutes on a thermal cycler, and placed on ice for 10 minutes. Ethanol precipitation was used to purify the reactions. The fragmented rRNA depleted RNA was combined with 5 μg of random hexamers (Roche) in a final volume of 11 μl, incubated at 65°C for 5 minutes, and afterwards immediately placed on ice. The remaining reagents were added to the reaction: 4 μl 5× First strand buffer (Invitrogen), 2 μl DTT (0.1 M Invitrogen), 1 μl dNTP mix (10 mM Invitrogen), 2 μl Actinomycin D (0.1 μg/ μl Sigma), 1 μl of RNase H (40U/ml Ambion), and 1 μl SuperScript III (200 U Invitrogen). The first strand reaction was incubated at 25°C for 10 minutes, 50°C for 90 minutes, followed by deactivation at 70°C for 15 minutes, followed by MinElute Reaction Cleanup Kit (Qiagen), according to the manufacturer's protocol. The second strand was synthesized by adding 20 μl 5x Second strand buffer (Invitrogen), 4 μl 5x First strand buffer (Invitrogen), 2 μl DTT (0.1 M Invitrogen), 1 μl random hexamers (5 mg/ml Roche), dUTP mix (12.5 mM Invitrogen), 1 μl RNase H (8U/ml Ambion), 1 μl E. coli DNA polymerase I (10U/ μl Invitrogen), and 1 μl E. coli DNA ligase (10 U/μl NEB) to the purified sample (100 μl total volume). After 2 hours at 16°C, 1 μl of T4 DNA polymerase (10U/μl Promega) was added and the reaction was incubated at 16°C for 10 minutes. The ds-cDNA was purified using the MinElute Reaction Cleanup Kit (Qiagen), according to the manufacturer's protocol. DNA samples were prepared for sequencing by end repair of 5 ng total cDNA 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 |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
HKC_L1L2_RNAseq_days0247_FPKM_genes.txt
HKC_L1L2_RNAseq_days0247_FPKM_isoforms.txt
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| Data processing |
42bp reads left after demultiplexing of original 50bp reads were mapped to the human genome NCBI build 37 (hg19) using gsnap version 2013-03-31.
Spliced reads were mapped based on the Ensembl 68 reference transcriptome.
Transcript quantification and differential expression analysis were done with cufflinks v2.1.1, using the RefSeq transcriptome (downloaded from UCSC genome browser website, June 2013).
Wiggle tracks were generated from read count-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: Tab-separated FPKM gene expression matrix containing RefSeq gene ID, HUGO gene symbol, genomic location and the FPKM values for the 8 samples.
Supplementary_files_format_and_content: Tab-separated FPKM isoform expression matrix containing RefSeq transcript ID, RefSeq gene ID, HUGO gene symbol, genomic location and the FPKM values for the 8 samples.
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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) |
| GSE59822 |
Transcription factor p63 bookmarks genomic loci in epithelial cells and regulates a subset of target genes during epidermal differentiation through dynamic enhancers (RNA-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 |
SAMN02943006 |
| SRA |
SRX663212 |
| Named Annotation |
GSM1446881_HKC_L1_RNAseq_day2.wig.gz |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1446881_HKC_L1_RNAseq_day2.wig.gz |
2.9 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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