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| Status |
Public on May 15, 2020 |
| Title |
RNA-seq_Primed-P3_rep4 |
| Sample type |
SRA |
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| Source name |
RNA-seq_Primed-P3
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| Organism |
Homo sapiens |
| Characteristics |
source cell type: human fibroblasts
cell type: reprogramming intermeidates or hiPSCs
cell subtype/time point: Primed-P3
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| 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.
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| 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.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 1500 |
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| Description |
E5_S22_L003
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| 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
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| 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
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| Phone |
+61 3 85598749
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| Organization name |
The University of Melbourne
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| Street address |
L 10, 305 Grattan Street
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| City |
Melbourne |
| State/province |
VIC |
| ZIP/Postal code |
3000 |
| Country |
Australia |
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| Platform ID |
GPL18460 |
| Series (1) |
| GSE149694 |
Transcriptional analysis of intermediates during human primed and naïve reprogramming. |
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| Relations |
| BioSample |
SAMN14790152 |
| SRA |
SRX8241220 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4509101_E5_S22_L003.txt.gz |
207.5 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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