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Sample GSM2044621

Query DataSets for GSM2044621

Status

Public on Jan 01, 2019

Title

cWAS-iMP1

Sample type

SRA

Source name

iPSC

Organism

Homo sapiens

Characteristics

individual: WAS patient 1
cell type: WAS-iPSC (gene corrected)
genotype: wild type
protocol: differentiated into macrophages

Growth protocol

iPSCs cultured on MEF feeders in human ESC medium were manually dissociated and plated on 8 days old overgrown OP9 feeders to start differentiation. Cells were co-cultured with the OP9 feeders for 12-14 days in OP9 differentiation medium (Alpha MEM containing 10% FBS, 100 μM monothioglycerol and 50 μg/ml ascorbic acid) as described by Vodyanik, M. A. & Slukvin, II. (Curr Protoc Cell Biol 2007). Differentiated cells were dissociated with collagenase and Accutase (Innovative Cell Technologies) and subjected to further differentiation into macrophages using GM-CSF and M-CSF.

Extracted molecule

total RNA

Extraction protocol

RNA was extracted with Qiagen RNeasy micro kit.
The Agilent TapeStation or Bioanalyzer was used to determine RNA integrity (RIN) numbers prior to library preparation. Stranded mRNA-Seq libraries were prepared using the TruSeq Stranded mRNA Library Prep Kit according to the manufacturer’s instructions (Illumina). Briefly, RNA with polyA tail was isolated using poly-T oligos conjugated to magnetic beads. mRNA was then fragmented and reverse-transcribed into cDNA. dUTPs were incorporated, followed by second strand cDNA synthesis. dUTP-incorporated second strand was not amplified. cDNA was then end-repaired, index adapter-ligated and PCR amplified. Purification of nucleic acids after each enzymatic steps was achieved by using AMPure XP beads (Beckman Coulter).
Strand specific polyA+ RNAseq (PE75)

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

human iPSC differentiated on OP9 feeders

Data processing

Illumina Casava v1.8.2 software used for basecalling.
For RNA-Seq, paired-end reads were aligned to the human genome (hg19) with STAR (v2.2.0c) with default parameters. Only uniquely alignable reads were used for downstream analysis.
RNA-Seq, Fragments Per Kilobase of exon per milion mapped reads were calculated using HOMER (http://homer.salk.edu/homer/) across annotated gene exons (RefSeq).
Genome_build: hg19
Supplementary_files_format_and_content: Tab-delimited text file containing gene expression values for each experiment. Columns definition, 1: Entrez Gene ID, 2: RefSeq ID, 3: chr, 4: start(hg19), 5: end(hg19), 6: strand, 7: transcript length, 8: Copies in genome, 9: Annotation/Symbol, 10-14: FPKM values, 15-23: additional annotations

Submission date

Jan 22, 2016

Last update date

May 15, 2019

Contact name

Christopher Benner

E-mail(s)

cbenner@ucsd.edu

Organization name

University of California, San Diego (UCSD)

Department

Medicine