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Status |
Public on Dec 08, 2020 |
Title |
BJOSKM96HS2 |
Sample type |
SRA |
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Source name |
newborn foreskin
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Organism |
Homo sapiens |
Characteristics |
cell line: BJ, ATCC cat# is CRL-2522 treatment: Transduced with the four reprogramming factors OSKM lentiviral preparation time: 96 hours post transduction growth protocol: grown in fibroblast media
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Treatment protocol |
fibroblasts were transduced with the reprogramming factors OCT4, SOX2, KLF4 and MYC. RNA was extracted at the indicated time post transduction (48 hours, 72 hours, and 96 hours). Lentiviral titers used were OCT4, 8; SOX2, 5; KLF4, 5; MYC, 3.
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Growth protocol |
human ESCs were grown in feeder-free/serum-free chemically defined E8 meium: E8 medium (pH 7.4) for iPSC maintenance: DMEM/nutrient mixture F-12 (F-12) (DMEM/F-12), 1.74 g/L NaHCO3, 1 g/L sodium chloride, 64 mg/L L-ascorbic acid 2-phosphate sesquimagnesium (see Note 6), 13.6 µg/L sodium selenium, 4 ng/mL FGF2, 20 μg/mL insulin, 10 μg/mL transferrin, and 2 μg/L TGFβ1 . Human fibroblasts were grown in fibroblast medium: Fibroblast growth medium: Dulbecco's Modified Eagle Medium (DMEM) with high glucose, supplemented with 10% heat-inactivated fetal bovine serum (FBS), 0.1 mM 2-mercaptoethanol, 1x penicillin-streptomycin (100 U/mL penicillin and 100 ug/mL streptomycin), 0.1 mM Minimum Essential Medium (MEM) Non-Essential Amino Acids (NEAA), and 4 ng/mL human basic fibroblast growth factor (bFGF, also known as FGF2)
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Extracted molecule |
polyA RNA |
Extraction protocol |
RNA was isolated using TRIzol™ Reagent (Invitrogen, Cat. 15596026) followed by DNA digestion with DNAseI (Thermo Fisher Scientific, Cat. AM2222) and a cleanup step using Quick-RNA Miniprep (Zymo Research, Cat. R1054). RNA concentration was determined using Nanodrop spectrophotometer (Thermo Fisher Scientific). Sequencing library was constructed by BGI using its protocol (Document #, SOP-SS-027; Version #, A0, Effective date: 2018-06-11)PolyA mRNA was purified from total RNA using the oligo(T) attached to magentic beads. The mRNA was then fragmented. cDNA was then generated using random hexamer-primerd reverse transcription followed by the second-strand cDNA synthesis. The synthesized cDNA was then subject to end-repair, and 3' adenylated. Adapters were then ligated to the ends of these 3' adenylated cDNA fragments. The cDNA was further PCR amplified. The amplified cDNA was purified with Ampure XP Beads. The library was validated on the Agilent 2100 bioanalyzer. The double strand PCR products were heat denatured and circularized by the splint oligo sequence. The single strand circular DNA were formated as the final library. RNA-seq using nanoball technology, sequening was conducted by BGI
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
DNBSEQ-G400 |
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Description |
Transduced with the four reprogramming factors OSKM lentiviral preparation, RNA extracted 96 hours post transduction
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Data processing |
Pre-alignment quality assessments of the raw fastq sequences were carried out using FastQC (version 0.11.7) (Andrews, 2010). The raw fastq sequences were aligned to the human hg38 reference genome (GenBank assembly accession: GCA_000001405.28). The alignments were carried out using STAR (version 2.7.1a) (Dobin et al 2013) with default parameters. Post-alignment quality assessments were carried out with RSeQC (version 2.6.3) (Wang et al 2012) and MultiQC (version 1.4) (Ewels et al 2016). Samtools (version 0.0.19) (Li et al 2009) and IGV (version 2.6.2) (Thorvaldsdóttir et al 2013) were used for indexing and viewing the alignments respectively. Gene expression was quantified as gene level counts using the htseq-count function (version 0.12.3) (Anders et al 2015); the Ensembl gene annotations for the human genome were used (genebuild-last-updated 2019-06). The htseq-count default parameters were used. Differentially expressed genes were identified using DESeq2 (version 1.28) (Love et al 2014). DESeq2 was run with default parameters (Love et al 2014). Genome_build: The human hg38 reference genome (GenBank assembly accession: GCA_000001405.28). Supplementary_files_format_and_content: excel file containing deseq2 normalized gene expression for each gene in each sample
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Submission date |
Oct 12, 2020 |
Last update date |
Dec 08, 2020 |
Contact name |
Kejin Hu |
Organization name |
UAB School of Medicine
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Department |
Department of Biochemistry and Molecular Genetics
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Lab |
Shelby Biomedical Research Building
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Street address |
1825 University Blvd.
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City |
Birmingham |
State/province |
AL |
ZIP/Postal code |
35294 |
Country |
USA |
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Platform ID |
GPL28038 |
Series (1) |
GSE159410 |
Human transcription factors responsive to initial reprogramming are predominantly legitimate during iPSC reprogramming |
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Relations |
BioSample |
SAMN16424226 |
SRA |
SRX9283444 |
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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