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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Mar 21, 2017 |
| Title |
mRNA Control ASO d20 rep1 |
| Sample type |
SRA |
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| Source name |
spinal cord
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| Organism |
Mus musculus |
| Characteristics |
tissue: spinal cord
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| Treatment protocol |
Mice were treated with PBS or Control or SMN ASO at d0 and sacrificed at d20 or d30.
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| Growth protocol |
SMA mice were generated as described in Staropoli et al., 2015.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
50-500ng DNase-treated total RNA was used to construct libraries using Illumina TruSeq Stranded mRNA HT Sample Prep Kit (Cat #RS-122-2103) with barcodes for sample multiplexing. The products were purified and enriched with PCR for 13 cycles to create final cDNA library. Libraries were quantified using HT DNA reagent kit (Part Number 760435, Caliper Life Sciences, Hopkinton, MA) using a LabChip GX (Perkin Elmer,Waltham,MA). Multiplexed libraries were equimolarly pooled, diluted to 2nM pool for final analysis on Agilent High sensitivity DNA kit to run on one rapid flowcell per lane and sequenced using 2x50 ntd paired end runs of the Illumina HiSeq 2500 platform.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
SMA_C1_SCT
mRNA-Seq
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| Data processing |
RNAseq reads were mapped with the STAR algorithm, aligning to the Gencode version M4 transcript annotation for mouse spinal cord RNA and the hg38 annotation for SH-SY5Y cells and hiPSC-MNs. We used parameters --alignIntronMin 20 --alignIntronMax 1000000 --outFilterMismatchNmax 999 --outFilterMismatchNoverLmax 0.05 --outFilterMultimapNmax 20 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --alignMatesGapMax 1000000 --sjdbOverhang 49. Aligned reads were assigned to transcripts from the annotations described above using RSEM, and resulting raw read counts were input into DESeq2 for differential gene expression analysis using default parameters.
For general splicing analysis, BAM files produced from STAR mapping were input into rMATS, using the Gencode version M4 transcript annotation for mouse spinal cord RNA and the hg38 annotation for SH-SY5Y and hiPSC-MN RNA samples. For detection of intron retention, novel human and mouse annotations were generated containing all consecutive spliced and unspliced exon-intron-exon triads from hg38 and Gencode vM4. To measure retention of U12 introns, a second set of annotations was generated by filtering this novel set of annotations for introns that overlapped exactly with human and mouse U12 introns extracted from U12DB. These annotations were used to call intron retention in mouse spinal cord, SH-SY5Y, and hiPSC-MN RNAseq datasets using rMATS. An FDR cutoff of 0.05 was used to define significantly differentially-spliced regions.
Genome_build: mRNA-seq: Gencode M4
Genome_build: DRIP-seq: hg38
Supplementary_files_format_and_content: SMArescue_all_RSEM.genes.results.human_names_split.txt: count matrix
Supplementary_files_format_and_content: SMArescue_d20d30_DvsE_all_DESeqOutput.txt: expression matrix
Supplementary_files_format_and_content: SMArescueIJKvsE.DESeq2ContrastTest.txt: expression matrix
Supplementary_files_format_and_content: SHSY5Y_shSMN-1_vs_shCtrl_FDR0.05.sepnames.txt: expression matrix
Supplementary_files_format_and_content: SHSY5Y_shSMN-2_vs_shCtrl_FDR0.05.sepnames.txt: expression matrix
Supplementary_files_format_and_content: SHSY5Y.RSEM.genes.results.human_names.txt: count matrix
Supplementary_files_format_and_content: hiPSC_shSMN-1_vs_shCtrl_FDR0.05.sepnames.txt: expression matrix
Supplementary_files_format_and_content: hiPSC_shSMN-2_vs_shCtrl_FDR0.05.sepnames.txt: expression matrix
Supplementary_files_format_and_content: hiPSC-MN.RSEM.genes.results.human_names.txt: count matrix
Supplementary_files_format_and_content: shCtrlrep1_S96_vs_IgG_summits_win250rep2.bed: peak
Supplementary_files_format_and_content: shSMNrep1_S96_vs_IgG_summits_win250rep2.bed: peak
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| Submission date |
Sep 22, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
John Carulli |
| Organization name |
Biogen
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| Department |
Computational Biology and Genomics
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| Lab |
Translational Genomics
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| Street address |
115 Broadway
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02142 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE87281 |
SMN deficiency in spinal muscular atrophy causes widespread intron retention and DNA damage |
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| Relations |
| BioSample |
SAMN05806579 |
| SRA |
SRX2186970 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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