 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Sep 01, 2015 |
| Title |
293 PTBP1 and PTBP2 double knockdown with exon-excluded PTBP1 overexpression_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
Flp-In™ T-REx™ 293 Cell Line
|
| Organism |
Homo sapiens |
| Characteristics |
genotype: inducible 3xFLAG exon-excluded PTBP1 at Flp-In locus
cell line: 293
|
| Treatment protocol |
siRNA transfection with Lipofectamine RNAiMax (Life Technologies)
|
| Growth protocol |
Monolayer growth in DMEM with 10% FBS
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was extracted from cultured cells using TRI reagent (Sigma).
Standard illumina protocol with DNAse treatment and polyA selection.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
293_all_splicing_events.tab
|
| Data processing |
Splicing analysis: Detailed description of the analysis pipeline can be found in [Irimia et al. Cell 159, 1511-1523 (2014)]. A summary of the main steps is provided here. An RNA-Seq dataset is first aliged to the genome using Bowtie. Reads that map to the genome are discarded from further analysis since exon-exon junctions (EEJs) are absent from genomic sequence. Reads that failed to align to the genome are aligned to a custom library of EEJs using Bowtie. To quantify splicing, the combination of EEJs that define each splicing event was created. For the case of single exon skipping events, we generated EEJs for C1A, AC2 and C1C2 (A represents the alternative exon and C1 and C2 represent the neighboring constitutive exons), requiring a minimum of eight positions from each exon. For multi-exon events we generated all possible forward combinations from C1 to C2 exons. If multiple alternative 5’ and/or 3’ splice sites were associated with any alternative, C1 or C2 exons, they were also included in the combinations. Read counts for each EEJ were then used to calculate the Percent Spliced In (PSI) of each event. A simplified format of the formula is: PSI = 100% * (reads supporting inclusion / total reads).
Genome_build: Homo sapiens, hg19; Gallus gallus, galGal3
Supplementary_files_format_and_content: 293_all_splicing_events.tab: tab delimited text file summarizing the splicing analysis of 293 data, where each row corresponds to a unique splicing event. The table has the following columns: GENE - gene ID, EVENT - unique event ID containing the ENSEMBL gene ID, COORD - genomic coordinates of the alternative exon, LENGTH - nucleotide length of the alternative exon, FullCO - genomic coordinates of alternative exon as well as the coordinates of the last and first basepairs of the upstream and downstream exons, respectively, COMPLEX - refers to type of splicing event (S/C1/C2/C3: alternative cassette exon > 15 nucleotides, ME: mutually exclusive exons, Alt3: alternative 3´ splice site, Alt5: alternative 5´ splice site, MIC: alternative micro cassette exon ≤ 15 nucleotides), siCon+siCon2 - PSI in cells treated with control siRNA (2 replicates), KD+KD2 - PSI in cells treated with PTBP1 + PTBP2 siRNA (2 replicates), Resc_L+Resc_L2 - PSI in cells treated with PTBP1 + PTBP2 siRNA with overexpression of full-length PTBP1 (2 replicates), Resc_S+Resc_S2 - PSI in cells treated with PTBP1 + PTBP2 siRNA with overexpression of exon-skipped PTBP1 (2 replicates), siCon.Q+siCon2.Q+KD.Q+KD2.Q+Resc_L.Q+Resc_L2.Q+Resc_S.Q+Resc_S2.Q - quality scores for the corresponding PSI values, see [Irimia et al. Cell 159, 1511-1523 (2014)] for details. // dt40_all_splicing_events.tab: tab delimited text file summarizing the splicing analysis of dt40 data, where each row corresponds to a unique splicing event. First 6 columns are the same as in 293_all_splicing_events.tab, WT1 + WT2 - PSI in wildtype cells (2 replicateS), HET34 + HET48 - PSI in cells with PTBP1 exon 8 delted from one allele (2 indepndent clones), HOM3 + HOM7 - PSI in cells with PTBP1 exon 8 deleted from both alleles (2 independent clones), WT1.Q+WT2.Q+HET34.Q+HET48.Q+HOM3.Q+HOM7.Q - quality scores for the corresponding PSI values, see [Irimia et al. Cell 159, 1511-1523 (2014)] for details.
|
| |
|
| Submission date |
Jun 08, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Serge Gueroussov |
| E-mail(s) |
serge.gueroussov@utoronto.ca
|
| Organization name |
University of Toronto
|
| Department |
Molecular Genetics
|
| Lab |
Dr. Benjamin Blencowe
|
| Street address |
160 College Street, Room 1030
|
| City |
Toronto |
| State/province |
Ontario |
| ZIP/Postal code |
M5S 3E1 |
| Country |
Canada |
| |
|
| Platform ID |
GPL16791 |
| Series (1) |
| GSE69656 |
An Alternative Splicing Event Amplifies Evolutionary Differences Between Vertebrates |
|
| Relations |
| BioSample |
SAMN03765114 |
| SRA |
SRX1053528 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
|
|
|
|
 |
