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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Apr 14, 2010 |
| Title |
-24 hours |
| Sample type |
SRA |
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| Source name |
Myoblast (exponential)
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| Organism |
Mus musculus |
| Characteristics |
cell line: C2C12
strain: C57BL/6
time point: -24 hours
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| Growth protocol |
Mouse skeletal muscle C2C12 cells were initially plated on 15 cm plates in DMEM with 20% fetal bovine serum. At confluence, the cells were switched to low serum medium to initiate myogenic differentiation. For extraction of total RNA, cells were first rinsed in PBS and then lysed in Trizol reagent (Invitrogen catalog # 15596-026) either during exponential growth in high serum medium, or at 60 hrs, 5 days and 7 days after medium shift. Residual contaminating genomic DNA was removed from the total RNA fraction using Turbo DNA-free (Ambion catalog # AM1907M). mRNA was isolated from DNA-free total RNA using the Dynabeads mRNA Purification Kit (Invitrogen catalog # 610-06).
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Preparation of cDNA followed the procedure described in Mortazavi et al. 2008 Nature Methods, with minor modifications as described below. RNA was fragmented to an average length of 200 nts by metal ion/heat catalyzed hydrolysis. The hydrolysis was performed in a 25 uL volume at 94°C for 90 seconds. The 5X hydrolyis buffer components are: 200 mM Tris acetate, pH 8.2, 500 mM potassium acetate and 150 mM magnesium acetate. After removal of hydrolysis ions by G50 Sephadex filtration (USA Scientific catalog # 1415-1602), the fragmented mRNA was random primed with hexamers and reverse-transcribed using the Super Script II cDNA synthesis kit (Invitrogen catalog # 11917010). After second strand synthesis, the cDNA went through end-repair and ligation reactions according to the Illumina ChIPSeq genomic DNA preparation kit protocol (Illumina catalog # IP102-1001), using the paired ends adapters and amplification primers (Illumina Catalog # PE102-1004). The cDNA library was size-fractionated on a 2% TAE low melt agarose gel (Lonza catalog # 50080), with a 100 bp ladder (Roche catalog # 14703220) run in adjacent lanes. Prior to loading on the gel, the ligated cDNA library was taken over a G50 Sephadex column to remove excess salts that interfere with loading the sample in the wells. After post-staining the gel in ethidium bromide, a narrow slice (~2mm) of the cDNA lane centered at the 300 bp marker was cut. The slice was extracted using the QiaEx II kit (Qiagen catalog # 20021), and the extract was filtered over a Microcon YM-100 microconcentrator (Millipore catalog # 42409) to remove DNA fragments shorter than 100 bps. Filtration was performed by pipeting the extract into the upper chamber of a microconcentrator, and adding ultra pure water (Gibco catalog # 10977) to a volume of 500 uLs. The filter was spun at 500 X g until only 50 uLs remained in the upper chamber (about 20 minutes per spin) and then the upper chamber volume was replenished to 500 uLs. This procedure was repeated 6 times. The filtered sample was then recovered from the filter chamber according to the manufacturer’s protocol. One-sixth of the filtered sample volume was used as template for 15 cycles of amplification using the paired-end primers and amplification reagents supplied with the Illumina ChIPSeq genomic DNA prep kit. The amplified product was then cleaned up over a Qiaquick PCR column (Qiagen catalog # 28104), and then the filtration procedure using the Microcon YM-30 microconcentrators described above was repeated, to remove both amplification primers and amplification products shorter than 100 bps. A final pass over a G50 Sephadex column was performed, and the library was quantified using the Qubit fluorometer and PicoGreen quantification reagents (Invitrogen catalog # Q32853). The library was then used to build clusters on the Illumina flow cell according to protocol.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer |
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| Description |
2x76bp RNA Seq
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| Data processing |
Alignment: Fragments were mapped to build 37.1 of the mouse genome using TopHat version 1.0.13 (http://tophat.cbcb.umd.edu)
Assembly and differential analysis: Transcripts were assembled and quantitated using Cufflinks 0.8.0 (http://cufflinks.cbcb.umd.edu)
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| Submission date |
Mar 11, 2010 |
| Last update date |
May 15, 2019 |
| Contact name |
Cole Trapnell |
| E-mail(s) |
cole@broadinstitute.org
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| Organization name |
Harvard University
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| Department |
Stem Cell and Regenerative Biology
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| Lab |
John Rinn
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| Street address |
7 Divinity Ave
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02138 |
| Country |
USA |
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| Platform ID |
GPL9185 |
| Series (1) |
| GSE20846 |
Transcript assembly and abundance estimation from RNA-Seq reveals thousands of new transcripts and switching among isoforms |
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| Relations |
| Reanalyzed by |
GSE80797 |
| SRA |
SRX017794 |
| BioSample |
SAMN00010202 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM521256_exp.gtf.gz |
5.1 Mb |
(ftp)(http) |
GTF |
| GSM521256_exp.sam.gz |
1.0 Gb |
(ftp)(http) |
SAM |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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