|
| Status |
Public on Oct 20, 2015 |
| Title |
MBNL_1_RNA-Seq |
| Sample type |
SRA |
| |
|
| Source name |
Neonatal Rat Cardiac Fibroblasts
|
| Organism |
Rattus norvegicus |
| Characteristics |
strain: Sprague-Dawley
|
| Treatment protocol |
To determine the mRNAs that directly bind to MBNL1 a Flag-epitope tagged MBNL1 was adenovirally overexpressed in cardiac fibroblasts. Fibroblast lysates were collected 36 hr later and then incubated with Flag-M2 affinity resin (Sigma) overnight at 4°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The resin was washed and RNA extracted using a Magna RIP kit (Millipore) and phenol-chloroform precipitation. For cardiac fibroblast experiments RNA was isolated and prepared with an RNAeasy kit (Qiagen).
Eluted RNAs were detected by RNAseq analysis using paired-end 100 nucleotide (nt) sequencing and 40 million reads for input-RNAs and RIP-RNAs. RNA Seq libraries were generated with Illumina TruSeq Stranded mRNA HT kits
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
MBNL1 murine cDNA adenoviral - Input RNA-Seq
|
| Data processing |
FASTQ sequence files were aligned to the rat genome version 5 using TopHat2 with de novo junction discovery.
Alternative splicing, alternative polyadenylation and differential gene expression analyses along with all visualized pathways, heatmaps and Venn 14 diagrams were obtained using AltAnalyze version 2.0.9 (Emig et al., 2010), and the Ensembl 77 / UCSC mRNA rn5 database filtering on genes with an RPKM>1. Alternative splicing events were identified using a reciprocal isoform percent spliced difference of at least 25% and putative alternative polyadenylation using a splicingindex difference > 2 or ASPIRE δI value > 0.2, occurring in the last of exon of the gene.
For all RNAseq experiments pathways and ontology enrichment analyses were performed with ToppGene (Chen et al., 2009) and GO-Elite (Zambon et al., 2012). All heatmaps from AltAnalyze were created using the HOPACH package via a connection to R (Salomonis et al., 2005).
Genome_build: rat genome version 5
|
| |
|
| Submission date |
Oct 20, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Nathan Salomonis |
| E-mail(s) |
nathan.salomonis@cchmc.org
|
| Organization name |
Cincinnati Children's Hospital
|
| Department |
Biomedical Informatics
|
| Lab |
Nathan Salomonis
|
| Street address |
3333 Burnet Avenue
|
| City |
Cincinnati |
| State/province |
OH |
| ZIP/Postal code |
45229 |
| Country |
USA |
| |
|
| Platform ID |
GPL18694 |
| Series (1) |
| GSE74185 |
MBNL1-mediated Regulation of Differentiation RNAs Promotes Myofibroblast Transformation and the Fibrotic Response |
|
| Relations |
| BioSample |
SAMN04196503 |
| SRA |
SRX1356548 |
