|
| Status |
Public on Jan 17, 2012 |
| Title |
CGN1 |
| Sample type |
SRA |
| |
|
| Source name |
cerebellar granular neurons (CGNs)
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57bl/6j
age: P8
growth: 16 hours in vitro
cell type: cerebellar granular neurons (CGNs)
|
| Growth protocol |
Cells were grown on a Poly-d-lysine (100 ug/mL) and Laminin (5 ug/mL) substrate
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Poly-A selection. RNA was prepared for Next Generation Sequencing following the Illumina mRNA Sample Preparation Guidelines (Illumina, Cat # RS-930-1001)
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Description |
Poly-A RNAs from P8 mice CGN neurons in culture
|
| Data processing |
Alignment: Tophat software with default settings except for the following options: –G option which supplies Tophat with gene model annotation (combined UCSC, Ensembl, and RefSeq annotations) and –i 50 which sets the minimum intron length to 50
Transcript reconstruction and expression estimation: Cufflinks software. At first Cufflinks uses the aligned reads in the dataset to describe a set of transcripts starting from the reads that span splice-junctions. We ran this step using a non-annotated reference genome because without an annotation the software will assemble novel transcripts and isoforms. After transcript assembly, normalized expression levels are estimated and reported as FPKM (Fragments Per Kilobase of exon per Million fragments mapped) together with confidence intervals. A different part of the software, named Cuffcompare (version 0.8.3) classified the reconstructed RNA-species as novel or known according to how they map back to the provided reference annotation. Cuffcompare was run twice, first with a combined reference GTF generated from crossing annotated transcripts found in the UCSC Genome, the Ensembl, and the RefSeq database. We combined three genome annotations in an effort to minimize falsely identified novel transcripts. UCSC was used as the base since it contained the highest number of annotated RNA species; all non overlapping annotations found in the other databases were added. We reran Cuffcompare in order to improve the accuracy of read alignment and therefore transcript expression estimation (personal communications with Cole Trapnell). The nature of the alignment of the reconstructed RNA-specie and the annotated element is reported according to a code letter. For our purposes we isolated from the output only the “=”, “j” and “u” classes (corresponding to a “perfect match” to a known RNA-molecule, new isoforms of known active locus, and to full transcripts never identified before ). We ran Cuffcompare a 2nd time after adding annotation for unknown and novel transcripts assembled by Cufflinks.
Differential expression test: Cuffdiff. Cuffdiff determined statistical significance based on the square root of the Jensen-Shannon divergence between the relative abundance of transcripts. Significance was reported as an uncorrected p-value, and then classified as significant/not –significant after Benjamini-Hochberg correction of the p-value.
Peak data (.bedgraph files) based on mm9 genome build.
|
| |
|
| Submission date |
Oct 31, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Dario Motti |
| Organization name |
Nationwide Children's Hospital
|
| Department |
Research
|
| Lab |
Kaspar
|
| Street address |
700 Childrens Drive
|
| City |
Columbus |
| State/province |
Ohio [OH] |
| ZIP/Postal code |
43205 |
| Country |
USA |
| |
|
| Platform ID |
GPL9250 |
| Series (1) |
| GSE33343 |
Isoform diversity and regulation in peripheral and central neurons revealed through RNA-Seq |
|
| Relations |
| Reanalyzed by |
GSE80797 |
| SRA |
SRX103964 |
| BioSample |
SAMN00744939 |
