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| Status |
Public on Oct 11, 2013 |
| Title |
RNASeq_Differentiated |
| Sample type |
SRA |
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| Source name |
Differentiated mouse ES cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: mixed differentiated cell population derived from mouse ES cells after 4.5 days in vitro differentiation induced by retinoic acid
passage: 29
strain: E14 [129P2/OlaHsd]
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| Growth protocol |
E14 mouse ES cells were grown in Dulbecco’s Modified Eagle’s Medium, High Glucose (DMEM, GIBCO® #11965-084), 15% Fetal Bovine Serum (FBS, HyClone #SH30071.03), 2 mM L-Glutamine (GIBCO® #25030-081), 0.1% 2-Mercaptoethanol (GIBCO® #21985-023) and 1000 u/ml ESGRO® supplement containing Leukemia Inhibitory Factor (Chemicon/Millipore #ESG1107), on mitomycin C treated mouse embryonic fibroblasts (MEF) plated on gelatinized cell culture plates. Prior to differentiation and to collection for RNA or chromatin preparation, ES cells were dissociated into single cell suspension using 0.25% Trypsin-EDTA (GIBCO® #25200056), and adsorbed twice, for 45 minutes each time, to remove MEF. Residual MEF contamination was estimated to be below 0.25% by visually inspecting using a hematocytometer. For ES cells differentiation, ES cells were plated at 10E4 cells/cm2 on gelatinized cell culture plates and grown in DMEM, 10% FBS, 2 mM glutamine, 0.1% 2-Mercaptoethanol and 0.1 µM all-trans-Retinoic acid (Sigma #R2625). After 4.5 days, cells were dissociated using 0.25 % Trypsin-EDTA and collected for chromatin or RNA preparation. In the cells collected for ChIP-seq and RNA-seq, cell death was estimated around 2% in undifferentiated cells and 8% in differentiated cells, using Trypan Blue staining.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted and purified using Trizol Reagent (Ambion®, #15596-026), according to manufacturer's instructions. The quality of total RNA was verified on a Bioanalyzer 2100 using the RNA 6000 Nano Total RNA kit (Agilent #5067-1511). Then, starting from 5 µg of total RNA, samples were prepared according to the Illumina mRNA sequencing sample preparation protocol (# RS-930-1001), with purification of ~250 bp cDNA templates from 2% agarose gel run at 100 V for 1 h. Two lanes were sequenced for each biological sample, with 36 bp single-end reads, on an Illumina Genome Analyzer IIx using Cluster Generation kits (v4) and Sequencing kits (v4).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Data processing |
We used a multi-step procedure to align the reads as previously described (Pal et al., Genome Res. 2011 Aug;21(8):1260-72.). In brief the following steps were taken. (1) Contamination removal – Reads matching to the mitochondrial genome sequence, ribosomal-like RNAs, and adapter sequences were removed. (2) Alignment to transcriptome – We generated a non-redundant combined transcriptome model using RefSeq, UCSC, Vega, Ensembl and MGI transcripts. The unaligned reads from the previous step were aligned to the combined transcriptome model. (3) Alignment to Aceview gene model – The unaligned reads from the previous step were mapped to the Aceview gene model (Thierry-Mieg and Thierry-Mieg, Genome Biol. 2006;7 Suppl 1:S12.1-14). (4) Alignment to genome – The unmapped reads from the previous steps were aligned directly to the genome. Reads were aligned allowing up to 2 mismatches and only the uniquely mapped reads were used for estimating expression.
Expression levels by transcript were calculated using the IsoformEx software (Kim et al., BMC Bioinformatics. 2011 Jul 27;12:305) and expressed as RPKM.
The expression for non-polyA tailed RNA class genes (miRNA, snoRNA, snRNA, rRNA, tRNA, scRNA) were removed from further analysis.
For transcripts sharing the same promoter, RPKM values were summed to obtain the expression by promoter. Transcripts were considered as originating from the same promoter if their first exon overlapped. Similarly, the expression for a given gene is the sum of the RPKM values of all the transcripts defining that gene.
Genome_build: mm9
Supplementary_files_format_and_content: The files "rnaseq_undifferentiated_lane1.wig" and "rnaseq_undifferentiated_lane2.wig" contain expression profiles in undifferentiated cells for sequencing lanes 1 and 2, respectively. The files "rnaseq_differentiated_lane1.wig" and "rnaseq_differentiated_lane2.wig" contain expression profiles in differentiated cells for sequencing lanes 1 and 2, respectively. The file "rnaseq_transcript_expression.tsv" indicates RPKM expression values by transcript, with col. 1-3, transcript location in mouse mm9 genome assembly; col. 4, transcript orientation; col. 5, transcript identifier(s); col. 6, corresponding gene name; col. 7, RPKM expression value in undifferentiated cells calculated from lane 1; col. 8, RPKM expression value in undifferentiated cells calculated from lane 2; col. 9, RPKM expression value in differentiated cells calculated from lane 1; col. 10, RPKM expression value in differentiated cells calculated from lane 2; col. 11, average RPKM expression value in undifferentiated cells, calculated from lanes 1 and 2; col. 12, average RPKM expression value in differentiated cells, calculated from lanes 1 and 2. The file "rnaseq_gene_expression.tsv" indicates RPKM expression values by gene, with col. 1-3, gene location in mouse mm9 genome assembly; col. 4, gene name; col. 5, gene orientation; col. 6, RPKM expression value in undifferentiated cells calculated from lane 1; col. 7, RPKM expression value in undifferentiated cells calculated from lane 2; col. 8, RPKM expression value in differentiated cells calculated from lane 1; col. 9, RPKM expression value in differentiated cells calculated from lane 2; col. 10, average RPKM expression value in undifferentiated cells, calculated from lanes 1 and 2; col. 11, average RPKM expression value in differentiated cells, calculated from lanes 1 and 2. The file "rnaseq_promoter_expression.tsv" indicates RPKM expression values by promoter, with col. 1-3, location in mouse mm9 genome assembly of the shared first exon between transcripts originating from the same promoter; col. 4, transcript(s) orientation; col. 5, corresponding gene name; col. 6, transcript(s) identifier(s); col. 7, RPKM expression value in undifferentiated cells calculated from lane 1; col. 8, RPKM expression value in undifferentiated cells calculated from lane 2; col. 9, RPKM expression value in differentiated cells calculated from lane 1; col. 10, RPKM expression value in differentiated cells calculated from lane 2; col. 11, average RPKM expression value in undifferentiated cells, calculated from lanes 1 and 2; col. 12, average RPKM expression value in differentiated cells, calculated from lanes 1 and 2.
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| Submission date |
Jul 20, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Sebastien Vigneau |
| E-mail(s) |
sebastien_vigneau@dfci.harvard.edu
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| Phone |
+1-857-540-5439
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| Organization name |
Dana-Farber Cancer Institute
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| Department |
Cancer Biology
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| Lab |
Alexander Gimelbrant
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| Street address |
450 Brookline Avenue
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL11002 |
| Series (2) |
| GSE39522 |
Differences in CTCF binding site sequence are associated with unique regulatory and functional trends during embryonic stem cell differentiation [RNA-Seq] |
| GSE39523 |
Differences in CTCF binding site sequence are associated with unique regulatory and functional trends during embryonic stem cell differentiation |
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| Relations |
| SRA |
SRX160970 |
| BioSample |
SAMN01091828 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM970850_rnaseq_differentiated_lane1.wig.gz |
37.8 Mb |
(ftp)(http) |
WIG |
| GSM970850_rnaseq_differentiated_lane2.wig.gz |
37.3 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Processed data are available on Series record |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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