 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jul 12, 2011 |
| Title |
A_AN19511_09 |
| Sample type |
SRA |
| |
|
| Source name |
postmortem brain tissue
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: brain region BA09 (frontal cortex)
sample status: Autism
|
| Treatment protocol |
NA
|
| Growth protocol |
NA
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Standard library preparation protocol for high throughput DNA sequencing on Illumina Genome Analyzer was performed at the Yale Center for Genome Analysis (detailed protocol at: http://medicine.yale.edu/keck/ycga/sequencing/protocols/index.aspx). Briefly, poly-A containing mRNA was purified using poly-T oligo-attached magnetic beads. Following purification, the mRNA was fragmented using divalent cations under elevated temperature. The cleaved RNA fragments were revers-transcribed using random primers, followed by second strand cDNA synthesis using DNA Polymerase I and RNaseH. cDNA fragments were then end-repaired by the addition of a single âAâ base, followed by ligation of the adapters. Finally, these products were purified and PCR-amplified to create the final cDNA library.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Description |
RNA-Seq
|
| Data processing |
We generate alternative splice junctions using sequences from three exons: C1 (upstream constitutively spliced exon), A (alternatively spliced exon) and C2 (downstream constitutively spliced exon). Quantification of alternative exon usage is determined by the percentage of reads aligning to inclusion junctions (C1-A and A-C2) relative to all junctions (C1-A, A-C2 and C1-C2).
Additional details: RNA sequencing and data analysis. 73-nucleotide reads were generated using an Illumina GAII sequencer according to the manufacturer's protocol. To generate sufficient read coverage for the quantitative analysis of alternative splicing events, reads for ASD and control brain samples were separately pooled and aligned to an existing database of EST and cDNA-derived alternative splicing junctions using the Basic Local Alignment Tool (BLAT) as described previously35, 36. Reads were considered properly aligned to a splice junction if at least 71 of the 73 nucleotides matched and at least 5 nucleotides mapped to each of the two exons forming the splice junction. Alternative exon inclusion values ('%inc'), representing the proportion of messenger RNA transcripts with the alternatively spliced exon included, were calculated for each mRNA pool as the ratio of reads aligning to the C1-A or A-C2 junctions against reads aligning against all three possible junctions as previously described35 (C1-A, A-C2, C1-C2, see Supplementary Fig. 3). Calculated %inc values were considered reliable if at least one of the included junctions as well as the skipped junctions were covered by at least 20 reads. %inc values were compared across samples using Fisher's exact test and the BonferroniâHochberg correction to identify differentially spliced exons associated with autism. Differential splicing events were considered significant if they fulfilled both criteria of FDR < 0.1 and %inc difference between autism and controls >15%.
|
| |
|
| Submission date |
Jul 12, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Xinchen Wang |
| E-mail(s) |
[email protected]
|
| Organization name |
MIT
|
| Street address |
32 Vassar St, D514
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02139 |
| Country |
USA |
| |
|
| Platform ID |
GPL9115 |
| Series (1) |
| GSE30573 |
Transcriptomic analysis of autistic brain reveals convergent molecular pathology [high-throughput sequence data] |
|
| Relations |
| SRA |
SRX083168 |
| BioSample |
SAMN00668207 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM758633_A_19511_09_processed.txt.gz |
252.7 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
|
|
|
|
 |
