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Sample GSM1608063

Query DataSets for GSM1608063

Status

Public on Jul 16, 2015

Title

GSM1282320_NT1

Sample type

SRA

Source name

Nuclear Transfer Stem Cell

Organism

Homo sapiens

Characteristics

cell type: Human Pluripotent Stem Cell
cell line: Nuclear Transfer Derived
sequenced molecule: polyA RNA

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

total 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

Description

re-accessioned sample

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
Supplementary_files_format_and_content: .txt file including DESeq2 normalized counts filtered for genes with at least 5 reads in at least one sample

Submission date

Feb 11, 2015

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