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Sample GSM4829257

Query DataSets for GSM4829257

Status

Public on Dec 08, 2020

Title

CRL48HS1

Sample type

SRA

Source name

newborn skin, male

Organism

Homo sapiens

Characteristics

cell line: CCD-1079Sk, ATCC Cat# is CRL-209
treatment: no treatment
time: 48 hours post seeding
growth protocol: grown in fibroblast media

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.

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)

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

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

DNBSEQ-G400

Description

RNA harvested 48 hours post seeding, no treatment

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

Submission date

Oct 12, 2020

Last update date

Dec 08, 2020

Contact name

Kejin Hu

Organization name

UAB School of Medicine

Department

Department of Biochemistry and Molecular Genetics

Lab

Shelby Biomedical Research Building