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Status |
Public on Jul 16, 2015 |
Title |
Sample19_Melas_iPS2_fib2 |
Sample type |
SRA |
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Source name |
Differentiated induced Pluripotent Stem Cell
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Organism |
Homo sapiens |
Characteristics |
cell type: Fibroblast cell line: Differentated iPSC sequenced molecule: polyA RNA
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Treatment protocol |
Sendai virus-based reprogramming was carried out using 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 as described previously (Ma, H. et al. Abnormalities in human pluripotent cells due to reprogramming mechanisms. Nature 511, 177-183, doi:10.1038/nature13551 (2014)). NT-ESC lines were produced from Leigh patient fibroblasts as described previously (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)). iPSC and NT-ESC lines were differentiated into fibroblasts as described previously (Paull, D. et al. Nuclear genome transfer in human oocytes eliminates mitochondrial DNA variants. Nature 493, 632-637, doi:10.1038/nature11800 (2013)).
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Growth protocol |
Leigh patient fibroblasts were acquired from Coriell Cell Repositories (catalogue no. GM13411) and cultured in DMEM/F12 with 10% fetal bovine serum (FBS). 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 as described previously (Ma, H. et al. Abnormalities in human pluripotent cells due to reprogramming mechanisms. Nature 511, 177-183, doi:10.1038/nature13551 (2014)). NT-ESC lines were produced from Leigh patient fibroblasts as described previously (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)). iPSC and NT-ESC lines were differentiated into fibroblasts as described previously (Paull, D. et al. Nuclear genome transfer in human oocytes eliminates mitochondrial DNA variants. Nature 493, 632-637, doi:10.1038/nature11800 (2013)).
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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.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2000 |
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Data processing |
RNA-Seq reads were trimmed and mapped to the GRCh37 reference using STAR (version 2.3.0.1). Four previously published IVF-ESC samples and four NT-ESC were added to increase statistical power and for quality control. 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 Ensembl GRCh37 refGene. Genes that did not have at least one sample with at least five reads were removed from the analysis. The data was normalized using the R (version 3.1.1) package DESeq2 (version 1.4.5). Eight samples were added for comparison: Four IVF-ESC samples: GSM1282324, GSM1378018,GSM1282325 and GSM1378019 and four NT-ESC samples: GSM1282320,GSM1378014, GSM1282321, GSM1378015. These have been re-accessioned and are included in this record. DESeq2 uses negative binomial generalized linear models and shrinkage estimation for dispersions and fold changes to improve stability and interpretability of the estimates. It reports a p-value and an adjusted p-value using the Benjamini-Hochberg procedure. 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) genome build: GRCH37 processed data files format and content: .txt file including DESeq2 normalized counts filtered for genes with at least 5 reads in at least one sample
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Submission date |
Sep 12, 2014 |
Last update date |
May 15, 2019 |
Contact name |
Robert E Morey |
E-mail(s) |
robmoreyucsd@gmail.com, remorey@health.ucsd.edu
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Organization name |
UCSD
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Department |
Pathology
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Lab |
Parast
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Street address |
2880 Torrey Pines Scenic Dr
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City |
La Jolla |
State/province |
CA |
ZIP/Postal code |
92037 |
Country |
USA |
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Platform ID |
GPL11154 |
Series (1) |
GSE61390 |
Genetic Correction and Metabolic Rescue of Pluripotent Cells from Patients with mtDNA |
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Relations |
BioSample |
SAMN03067503 |
SRA |
SRX700354 |
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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