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GEO help: Mouse over screen elements for information. |
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| Status |
Public on May 21, 2011 |
| Title |
TT2_WT_mRNA-seq |
| Sample type |
SRA |
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| Source name |
Embyonic Stem Cell
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| Organism |
Mus musculus |
| Characteristics |
cell type: Embyonic Stem Cell
strain: TT2 (C57BL/6/CBA F1)
genotype/variation: WT
chip antibody: none
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| Treatment protocol |
none
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| Growth protocol |
J1 and TT2 ES cells were passaged every 48-72 hours in DMEM supplemented with 15% FBS (HyClone), 20mM HEPES, 0.1mM nonessential amino acids, 0.1mM 2- mercaptoethanol, 100 units/ml penicillin, 0.05 mM streptomycin, leukemia inhibitory factor (LIF) and 2mM glutamine on gelatinized plates.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
RNA was extracted using TRIzol following the manufactures instructions and DNaseI treated (Invitrogen, Carlsbad, CA, USA). PolyA+ RNA was purified using the MACS mRNA isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) from 10ug of DNaseI-treated total RNA as per manufacturer?s instructions. mRNA-seq libraries were constructed from purified polyA RNA, as described in Morin et al. (Morin et al., 2008), from 10ug of DNAse1 treated total RNA and sequenced on an Illumina Genome AnalyzerIIx following the manufactures recommended protocol (Illumina Inc., Hayward, CA).
Sheared cDNA or IPed material was resolved on a 12% acrylamide gel and the 100-300bp size fraction was eluted from the gel slice overnight at 4oC in 300 ml of elution buffer (5:1, LoTE, 7.5M Ammonium Acetate). DNA was recovered from the resulting gel slurry using a QIAquick PCR Purification Kit (Qiagen). Following column elution, the DNA ends were repaired using a 1:5 mixture of T4 and Klenow DNA polymerases (New England Biolabs), purified by phenol chloroform isoamyl alcohol (pH 8.0, 100ul, Fisher) extraction and EtOH precipitated. A single "A" base was added to the DNA fragments using Klenow exo- (3' and 5' exo minus) (New England Biolabs). Adapters (Illumina) were ligated to ends of the "A"-tailed DNA and adapter-modified DNA fragments enriched by PCR using Phusion polymerase (Finnzymes) and PCR primer 1.1 and 2.1 (Illumina) following the manufacturer's instructions (15 PCR cycles). The resulting PCR product was purified using a 8% PAGE (Nuvex; Invitrogen, Burlington, ON, Canada) to remove small products including adapter dimers, and the DNA quality assessed using an Agilent DNA 1000 series II assay and quantified by Qubit fluorometer (Invitrogen, Burlington, ON, Canada) and then diluted to 10nM. Cluster generation and 51 or 76 cycles of sequencing were performed on the Illumina cluster station and Illumina Genome AnalyzerIIx sequencing platform
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
Non-directional mRNA-seq
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| Data processing |
Alignment (RNA-seq): Sequence reads were aligned to the mouse reference genome (mm9) using MAQ v0.7.1 (Li et al., 2008), with Smith-Waterman alignment disabled and annotated exon-exon junctions compiled from Ensembl v54, RefSeq and UCSC gene annotation sources (downloaded from http://genome.ucsc.edu on March 17, 2009), as described (Morin et al., 2010; Shah et al., 2009). Sequence reads that could be uniquely assigned a position in the transcript resource (exon-exon junctions) were computationally repositioned to the genomic mm9 coordinates and a single merged bam file (Li et al., 2009) generated for downstream analyses.
Wigs (RNA-seq):The Samtools pileup utility (Li et al., 2009) and FindPeaks 3.1 (Fejes et al., 2008) was used to generate data tracks for visualization (wig and bigWig (Kent et al., 2010)) in the UCSC browser. Repbase (Jurka et al., 2005), a comprehensive database of repetitive elements, was used for alignment of reads to repetitive elements.
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| Submission date |
May 20, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Eric Chuah |
| E-mail(s) |
echuah@bcgsc.ca
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| Phone |
604-707-5900 3231
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| Organization name |
BC Genome Sciences Centre
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| Street address |
570 West 7th Ave
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| City |
Vancouver |
| State/province |
BC |
| ZIP/Postal code |
V5Z 4S6 |
| Country |
Canada |
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| Platform ID |
GPL9250 |
| Series (1) |
| GSE29413 |
DNA methylation and SETDB1/H3K9me3 regulate predominantly distinct sets of genes, retroelements and chimaeric transcripts in mouse ES cells |
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| Relations |
| Reanalyzed by |
GSE80797 |
| SRA |
SRX063936 |
| BioSample |
SAMN00619174 |
| Named Annotation |
GSM727423.wig.gz |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM727423.bam |
1.2 Gb |
(ftp)(http) |
BAM |
| GSM727423.wig.gz |
33.9 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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