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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Feb 02, 2017 |
| Title |
RNA-Seq of N2A cells after non-targeting knockdown (control for Srrm4 and Zfp871 knockdowns), replicate 1 |
| Sample type |
SRA |
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| Source name |
Neuroblastoma cells
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| Organism |
Mus musculus |
| Characteristics |
cell line: N2A
knockdown: siNT
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| Treatment protocol |
Cells were transfected with SMARTpool siRNAs (Dharmacon) using Lipofectamine RNAiMAX (Invitrogen), as recommended by the manufacturer. A non-targeting siRNA pool was used as a control. Cells were harvested 48 hours post transfection.
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| Growth protocol |
Mouse neuroblastoma (N2A) cells were grown in DMEM supplemented with 10% FBS, sodium pyruvate, MEM non-essential amino acids, and penicillin/streptomycin, and maintained at 37°C with 5% CO2.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was extracted either with TriZol (ThermoFisher) or the Qiagen RNasy kit.
RNA libraries were prepared for sequencing using standard Illumina protocols
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
vast-tools.AltSplicing_Srrm4.Zfp871.tab
AltSplicing_Srrm4.Zfp871.tab
Expression_Srrm4.Zfp871.tab
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| Data processing |
CASAVA 1.8 was used for base calling.
RNA-seq reads were processed for AS and expression analysis using our pipeline vast-tools, version 1.0 (Braunschweig et al., Genome Res. (2014); Irimia et al., Cell (2014)), which is available on github (https://github.com/vastgroup/vast-tools).
Alternative splicing analysis: From primary vast-tools output, events with poor coverage or junction balance were filtered out (vast-tools quality column score 3 other than SOK/OK/LOW for cassette exon [CE], microexon [MIC] and alternative 5'/3'-ss events or coverage less than 15 reads for intron retention [IR] events; score 4 other than OK/B1 for CE and MIC events or less than 0.05 for IR events). Significantly differential AS was assessed with the diff module of vast-tools under development and were additionally required to have a PSI difference >10.
Expression analysis: Expression differences were calculated based on vast-tools raw read counts per gene. In cases with a single replicate (knockdown of Nacc1 and Mbnl1/Mbnl2), counts were converted to read-per-million (RPM) and changes calculated as log2((1 + RPM[siSpecific]) / (1 + RPM[siNT])), and genes were required to have a vast-tools cRPKM ≥3 and a raw count of ≥10 in at least one of the compared samples. In cases with multiple replicates (knockdown of Srrm4 and Zfp871), differential expression was assessed with the R package edgeR.
Genome_build: mm9
Supplementary_files_format_and_content: All files are in tab-delimited format. Files vast-tools.AltSplicing_Mbnl.Nacc1.tab and vast-tools.AltSplicing_Srrm4.Zfp871.tab contain primary vast-tools output. Files AltSplicing_Mbnl.Nacc1.tab and AltSplicing_Srrm4.Zfp871.tab contain filtered PSI values and annotations of differential AS. Files Expression_Mbnl.Nacc1.tab and Expression_Srrm4.Zfp871.tab each contain raw counts per gene as well as fold changes and associated p-values, where applicable.
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| Submission date |
Apr 12, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Ulrich Braunschweig |
| Organization name |
University of Toronto
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| Department |
Donnelly Centre
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| Lab |
Benjamin J. Blencowe
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| Street address |
160 College Street
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| City |
Toronto |
| State/province |
Ontario |
| ZIP/Postal code |
M5S 3E1 |
| Country |
Canada |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE80204 |
Multilayered control of alternative splicing regulatory networks by transcription factors (RNA-Seq) |
| GSE80205 |
Multilayered control of alternative splicing regulatory networks by transcription factors |
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| Relations |
| BioSample |
SAMN04687430 |
| SRA |
SRX1700546 |
| 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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