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Sample GSM4509089 Query DataSets for GSM4509089
Status Public on May 15, 2020
Title RNA-seq_5iLAF-D21_rep2
Sample type SRA
 
Source name RNA-seq_5iLAF-D21
Organism Homo sapiens
Characteristics source cell type: human fibroblasts
cell type: reprogramming intermeidates or hiPSCs
cell subtype/time point: 5iLAF-D21
Growth protocol To generate human primed and naïve iPSCs used for this study, somatic cell reprogramming with the Cytotune 2.0 kit, experiments were performed according to the manufacturer’s instructions (Invitrogen). Briefly, human dermal fibroblast (adult) were seeded at ~5x104 cells per well of a 6-well plate in fibroblast medium containing DMEM (Gibco), 10% FBS (Hyclone), 1% nonessential amino acids (Gibco), 1mM GlutaMAX (Gibco), Pen-strep (Gibco), 0.1mM 2-mercaptoethanol (Gibco) and 1mM sodium pyruvate (Gibco) . After 2 days, cells were transduced with Sendai viruses in fibroblast medium with multiplicity of infections (MOIs) as follows, KOS MOI=5, c-MYC MOI=15, KLF4 MOI=6. After 24 hours, the medium was replaced with fresh fibroblast medium and media were changed every other day thereafter. On day 7, cells were trypsinized and seeded onto a layer of iMEF feeders at ~0.5x105~1x105 per flask in fibroblast medium. The next day, the cells were switched to primed and a few types of naive media. 21-24 days after transduction, cells were passaged and expanded. Reprogramming intermediates were collected at the indicated time.
Extracted molecule total RNA
Extraction protocol Reprogramming intermediates and hiPSCs were isolated by FACS (Supplementary Table 4) and RNA extraction was performed using the RNeasy micro kit (Qiagen, Cat#74004) from ∼2–20 × 10^4 cells with QIAcube (Qiagen). The concentrations of RNA were measured by a Qubit RNA HS Assay Kit (ThermoFisher, Cat#Q32855) on a Qubit 2.0 Fluorometer (ThermoFisher).
~25 ng of RNA was used for library construction with the SPIA kit (NuGen) and subsequently sequenced by HiSeq 1500 or HiSeq 3000 sequencer (Illumina). Sequencing libraries were single-end with 50 nt length and a targeted number of reads of 20-30 million.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 1500
 
Description 15-00019_AAGAGG_L0012
Data processing Cellranger mkref (v2.1.0) was run to create a reference using the modified GTF annotation file and the hg19 genome (GRCh37, CellRanger reference version 1.2.0, genome build GRCh37.p13, which contained the Sendai virus KLF4, MYC and SeV sequences as extra chromosomes, also accounted for as extra annotations on the annotation file).
Low quality sequencing reads and were filtered and trimmed with Trimmomatic (v 0.36, Phred score of 6 consecutive bases below 15, minimum read length of 36nt) and mapped to a custom version of hg19 human genome (with modifications described above with STAR (v 2.4.2a).
Gene read counting was performed with featureCounts (v1.5.2, unstranded) against the custom version of Ensembl’s GRCh37 annotation with modifications described above.
Genome_build: hg19
Supplementary_files_format_and_content: Each sample gene (rows) read counts matrices
 
Submission date May 01, 2020
Last update date May 15, 2020
Contact name Fernando Rossello
E-mail(s) frossello@unimelb.edu.au, fernando.rossello@monash.edu
Phone +61 3 85598749
Organization name The University of Melbourne
Street address L 10, 305 Grattan Street
City Melbourne
State/province VIC
ZIP/Postal code 3000
Country Australia
 
Platform ID GPL18460
Series (1)
GSE149694 Transcriptional analysis of intermediates during human primed and naïve reprogramming.
Relations
BioSample SAMN14790225
SRA SRX8241208

Supplementary file Size Download File type/resource
GSM4509089_15-00019_AAGAGG_L0012.txt.gz 208.0 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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