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| Status |
Public on Jan 01, 2014 |
| Title |
WT MEFs -1 |
| Sample type |
SRA |
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| Source name |
embyronic fibroblasts
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| Organism |
Mus musculus |
| Characteristics |
genotype: WT
strain: c57/b6
cell type: MEF
passage: 2
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| Growth protocol |
Parental Nanog-/- mESCs were obtained from I. Chambers and were cultured on feeders in 2i + LIF conditions as previously described(Chambers et al., 2007; Silva et al., 2008). To obtain Nanog-/- MEFs, Nanog-/- mESCs were injected into blastocyst stage embryos. At E12.5 MEFs were dissected out and sorted for constitutive GFP expression, indicating Nanog-/- genotype (Chambers et al., 2007). For reprogramming, MEFs were transduced with retroviruses carrying murine Oct4, Sox2, Klf4, +/- c-Myc exactly as described by Ichida et al (Ichida et al., 2009). On day 20 post-transduction with reprogramming transgenes, iPS colonies were picked and passaged onto feeders and cultured in 2i+LIF conditions with passaging every 5 days (Silva et al., 2008). MEFs were grown in 10% FBS.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was harvested using Trizol (Invitrogen) according to the manufacturers’ directions. RNA quality was determined using BioAnalyzer (Aligent). RNA integrity numbers above 7.5 were deemed sufficiently high quality to proceed with library preparation
RNA sequencing libraries were generated from ~250 ng total RNA using the illumina TruSeq RNA kit v2, according to the manufacturers' directions. Libraries were sequenced at the Broad Institute genomics platform on a HiSeq 2500 or at the Harvard Northwest labs sequencing facility on a HiSeq 2000.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
JI_6
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| Data processing |
Basecalls performed using CASAVA
Reads were aligned to the genome using the split read aligner TopHat (v2.0.7) and Bowtie2 (v2.0.5) using default parameters. Reference files of the murine genome build mm10, as well as ensembl transcript annotations, were obtained from iGenomes (http://support.illumina.com/sequencing/sequencing_software/igenome.ilmn, March 2013 build).
Differential expression was performed using Cuffdiff (v2.1.1) using default parameters and flags —min-reps-for-js-test 2, GTF from iGenomes was supplied for transcript assembly.
Genome_build: mm10 : ensembl compatible from igenomes (NCBIM37) March 2013 update
Supplementary_files_format_and_content: The FPKM values calculated by cuffdiff are reported as tab-delimited files for each sample (FPKM.txt). FPKMs calculated across replicates as well as confidence intervals are provided in the FPKM_matrix.txt file.
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| Submission date |
Dec 11, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Brandi Nicole Davis-Dusenbery |
| Organization name |
Harvard University
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| Department |
SCRB
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| Lab |
Kevin Eggan Laboratory
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| Street address |
7 Divinity Ave
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| City |
Cambridge |
| State/province |
Massachusetts |
| ZIP/Postal code |
02125 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE53212 |
Nanog Independent Reprogramming to iPSCs with Canonical Factors |
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| Relations |
| BioSample |
SAMN02440164 |
| SRA |
SRX390154 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1287749_JIfib_0_FPKM.txt.gz |
386.7 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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