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| Status |
Public on Jul 18, 2013 |
| Title |
TSW491 |
| Sample type |
SRA |
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| Source name |
R6/2 wildtype littermate, striatum, 12 weeks
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| Organism |
Mus musculus |
| Characteristics |
strain/background: C57Bl/6 / CBA
genotype/variation: wild type
tissue: brain striatum
age: 12 weeks
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| Extracted molecule |
total RNA |
| Extraction protocol |
Flash-frozen tissues were homogenized and RNA extracted with TRIzol Reagent and purified with RNeasy columns (Qiagen), all samples with RINs greater than 7. Oligo d(T)25 Magnetic Beads (New England BioLabs) were used to isolate mRNA from 1-3μg of total RNA. Purified mRNA was fragmented with Ambion's RNA fragmentation kit for 5 minutes at 70°C.
Library construction was adapted from the dUTP strand-specific protocol of Levin et al. 2010. mRNA was ethanol precipitated, concentrated in 5μl of water, and pre-mixed with 3μg of random hexamers for 5 minutes at 65°C before chilling on ice. First-strand cDNA was synthesized as described, but incubated as follows: 10 minutes at 25°C, 50 minutes at 42°C, and 15 minutes at 70°C. First-strand cDNA was purified by PCI extraction, ethanol precipitated with 0.1 volume 3M ammonium acetate to remove dNTPs, and resuspended in 104μl H2O. Second-strand cDNA was synthesized as described, but incubated for 2.5 hours. Paired-end libraries for Illumina sequencing were then prepared from the cDNA as previously published except adaptor-ligated cDNA was size-selected to 200-400bp, and we performed PCR using Phusion High-Fidelity DNA polymerase with HF buffer (New England BioLabs) and 10ul of Q Solution (Qiagen). These paired-end, strand-specific cDNA libraries were then sequenced on the Illumina Genome Analzyer (36 bp reads) or HiSeq (40 bp reads).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Description |
Strand-specific paired-end mRNA-Seq.
Summary processed data file: TSrpkms.txt
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| Data processing |
Reads were mapped to mm9 with the Bowtie alignment program with settings -m 1 -v 2 --best.
Differentially expressed genes were identified with the R package DESeq, using a 10% FDR cutoff and log2 difference of 0.5 between wild-type and mutant conditions.Outliers were further excluded by restricting the residual variance quotients to less than 10.
Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated.
Genome_build: MGSCv37
Supplementary_files_format_and_content: RPKM files are tab-delimited text files with the following four fields: NCBI Gene IDs, raw counts, #positions in gene, and RPKMs. Summary text files list the RPKMs for each sample in a condition (8wk cortex, 8wk striatum, 12wk cortex, 12wk striatum).
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| Submission date |
Jul 17, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Christopher Ng |
| Organization name |
Massachusetts Institute of Technology
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| Street address |
77 Massachusetts Ave.
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| City |
Cambridge |
| State/province |
Massachusetts |
| ZIP/Postal code |
02139 |
| Country |
USA |
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| Platform ID |
GPL11002 |
| Series (2) |
| GSE48962 |
Targeting H3K4 methylation as a therapeutic strategy for Huntington's disease (RNA-seq) |
| GSE48963 |
Targeting H3K4 methylation as a therapeutic strategy for Huntington's disease |
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| Relations |
| BioSample |
SAMN02256576 |
| SRA |
SRX323809 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1187672_TSW491.combined.rpkm.gz |
230.6 Kb |
(ftp)(http) |
RPKM |
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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