 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jul 08, 2014 |
| Title |
Sample11_HDF |
| Sample type |
SRA |
| |
|
| Source name |
Human Dermal Fibroblast
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Fibroblast
cell line: Parental
|
| Treatment protocol |
Fetal origin human dermal fibroblasts (HDF) were acquired from ScienCell Research Laboratories (catalog # 2300) and cultured in DMEM/F12 with 10% fetal bovine serum (FBS). Genetically matched iPSCs were generated by transductions of the same HDF culture with retroviral or Sendai virus vectors carrying OCT4, SOX2, KLF4, and MYC. Sendai virus-based reprogramming was carried out according to the manufacturer’s protocol (CytoTune™-iPS Reprogramming Kit, Life Technologies, Inc). Colonies with typical morphology were isolated and manually propagated similar to NT-ESC and IVF-ESC protocols.
|
| Growth protocol |
Fetal origin human dermal fibroblasts (HDF) were acquired from ScienCell Research Laboratories (catalog # 2300) and cultured in DMEM/F12 with 10% fetal bovine serum (FBS). Cells were transduced by retro virus-based iPSC vectors as previously reported (Lowry, W. E. et al. Generation of human induced pluripotent stem cells from dermal fibroblasts. Proceedings of the National Academy of Sciences of the United States of America 105, 2883-2888, doi:10.1073/pnas.0711983105 (2008)). Sendai virus-based reprogramming was carried out according to the manufacturer’s protocol (CytoTune™-iPS Reprogramming Kit, Life Technologies, Inc). Colonies with typical morphology were isolated and manually propagated similar to NT-ESC and IVF-ESC protocols (Tachibana, M. et al. Human embryonic stem cells derived by somatic cell nuclear transfer. Cell 153, 1228-1238, doi:10.1016/j.cell.2013.05.006 (2013)).
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
RNA was isolated (TRIzol Reagent, Life Technologies, Inc.) quantified (Qubit RNA Assay Kit, Life Technologies, Inc.) and quality controlled (RNA6000 Nano Kit and BioAnalyzer 2100, Agilent).
Approximately 500 ng was used as input for the Illumina TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, Inc.) and sequencing libraries were created according to the manufacturer’s protocol. Briefly, poly-A containing mRNA molecules were purified using poly-T oligo-attached magnetic beads. Following purification, the mRNA was fragmented and copied into first strand cDNA using random primers and reverse transcriptase. Second strand cDNA synthesis was then done using DNA Polymerase I and RNase H. The cDNA was then ligated to adapters and enriched with PCR to create the final cDNA library. The library was then pooled and sequenced on a HiSeq 2000 (Illumina, Inc.) instrument as per manufacturer’s instructions. Sequencing was performed up to 2 X 101 cycles.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
RNA-Seq reads were trimmed and mapped to the hg19 reference using STAR (version 2.3.0.1)
Samtools (version 0.1.17) was then used to sort, merge, and eliminate duplicate reads
Expression levels for each gene were quantified using the python script rpkmforgenes and annotated using RefSeq (archive-2012-03-09-03-24-410). Genes that did not have at least one sample with at least ten reads were removed from the analysis.
The data was normalized using the R (version 3.0.1) package DESeq (version 1.12.0) and batch corrected using the R script ComBat. Batch correction can create negative values.
Differential expression analysis was done using ANOVA in Qlucore Omics Explorer 2.3
Transcripts with an adjusted P-Value less than 0.05 were considered differentially expressed.
All enrichment analysis was performed using GREAT (v. 2.0.2)8 with default settings.
genome build: hg19
processed data files format and content: txt file including DESeq normalized counts filtered for genes with at least 10 reads in at least one sample
|
| |
|
| Submission date |
Dec 06, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Robert E Morey |
| E-mail(s) |
robmoreyucsd@gmail.com, remorey@health.ucsd.edu
|
| Organization name |
UCSD
|
| Department |
Pathology
|
| Lab |
Parast
|
| Street address |
2880 Torrey Pines Scenic Dr
|
| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
| |
|
| Platform ID |
GPL11154 |
| Series (2) |
| GSE53094 |
Epigenetic and transcriptional aberrations in human pluripotent stem cells reflect differences in reprogramming mechanisms [RNA-Seq] |
| GSE53096 |
Epigenetic and transcriptional aberrations in human pluripotent stem cells reflect differences in reprogramming mechanisms |
|
| Relations |
| BioSample |
SAMN02438041 |
| SRA |
SRX388463 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
|
|
|
|
 |
