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| Status |
Public on Jul 15, 2016 |
| Title |
RNA-Seq Undifferentiated EpSC Replicate 2 |
| Sample type |
SRA |
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| Source name |
Undifferentiated EpSC
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| Organism |
Homo sapiens |
| Characteristics |
tissue: foreskin
cell type: Epidermal Stem Cells (EpSC)
passage: 2
cell: Primary
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| Treatment protocol |
For calcium-induced differentiation, after reaching 70% confluence KSFM was exchanged for EMEM (Lonza) supplemented with 8% chelated FBS, EGF (10 ng/ml), 1% penicillin/streptomycin, and 1.2mM CaCl2. After 48 hr differentiated keratinocytes were collected for analysis
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| Growth protocol |
Human epidermal stem cells were isolated independently from three neonatal foreskin samples and cultured with a feeder layer of fibroblasts (J2-3T3) as described previously (Janich et al., Cell Stem Cell, 2013). Undifferentiated keratinocytes were grown in Keratinocyte Serum-Free Medium with supplements as indicated by the manufacturer (KSFM; GIBCO).
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using TRIZOL (LifeTechnologies) and cleaned up through RNA binding affinity columns (Quiagen) following manufacturer instructions
The library from total RNA from three biological replicates was prepared using the TruSeq®Stranded Total Sample Preparation kit (Illumina Inc.,) according to manufacturer’s protocol. Briefly, rRNA was depleted from 0.5 ug of total RNA using the Ribo-Zero Gold Kit followed by fragmentation by divalent cations at elevated temperature resulting into fragments of 80-450nt, with the major peak at 160nt. First strand cDNA synthesis by random hexamers and reverse transcriptase was followed by the second strand cDNA synthesis, performed in the presence of dUTP instead of dTTP. Blunt-ended double stranded cDNA was 3´adenylated and the 3´-“T” nucleotide at the Illumina indexed adapters was used for the adapter ligation. The ligation product was amplified with 15 cycles of PCR. Each library was sequenced using TruSeq SBS Kit v3-HS, in paired end mode with the read length 2x76bp. We generated minimally 137 million paired end reads for each sample run in one sequencing lane on HiSeq2000 (Illumina, Inc) following the manufacturer’s protocol. Images analysis, base calling and quality scoring of the run were processed using the manufacturer’s software Real Time Analysis (RTA 1.13.48) and followed by generation of FASTQ sequence files by CASAVA.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
processed data file: RNAseq_UNDIFF_DIFF_fpkm.txt
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| Data processing |
The RNA-seq datasets were aligned to the human genome build hg19 using the GEM split mapper version 7.0 (Marco-Sola et al., 2012) with default parameters.
To quantify the gene and transcript expression levels, Cufflinks (version 2.1.1) (Trapnell et al., 2010) was run with UCSC hg19 reference annotations, providing FPKM values.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited txt file containing FPKM values
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| Submission date |
Mar 16, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Debayan Datta |
| Organization name |
IRB Barcelona
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| Department |
Oncology
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| Lab |
Stem Cells and Cancer
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| Street address |
C. Baldiri Reixac 10
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| City |
Barcelona |
| State/province |
Barcelona |
| ZIP/Postal code |
08028 |
| Country |
Spain |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE65838 |
Dnmt3a and Dnmt3b associate with enhancers to regulate human epidermal stem cell homeostasis |
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| Relations |
| BioSample |
SAMN03418090 |
| SRA |
SRX957243 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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