|
| Status |
Public on Aug 20, 2019 |
| Title |
Dex-treated (24hrs) X2 (duplicate) |
| Sample type |
SRA |
| |
|
| Source name |
Neonatal Rat Ventricular Myocytes
|
| Organism |
Rattus norvegicus |
| Characteristics |
strain: Sprague Dawley
cell type: Neonatal Rat Ventricular Myocytes
treatment: Dex-treated (24hrs)
|
| Treatment protocol |
Cardiomyocytes are treated with Dexamethasone (Sigma) at concentration of 100nM or Ethanol (control)
|
| Growth protocol |
Neonatal Rat Ventricle myocytes are cultured from 1day old SD rat pups by colageanase and DNAse digestion. Cardiomyocytes are enriched by Percoll gradient and preplating, which removes non-cardiomyocytes. Cardiomyocytes are plated in medium (DMEM-F12, containing high glucose, L-glutamine and HEPES) with 10% FBS. Cardiomyocytes are changed to serum-free medium after 24hrs.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cardiomyocytes are scraped and snapped frozen in liquid nitrogen and stored at -140degreeC. Cells were sent to Active Motif for RNAseq.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
RNAseq data analysis was done by Active Motif. Data analysis report is attached as Pdf
Directional Poly-A RNA-seq libraries were prepared and sequenced as PE75 (75-bp paired-end reads) on Illumina NextSeq 500 to a depth of 47-53M read pairs. The “TopHat” algorithm was used to align the reads to the rn5 genome: http://ccb.jhu.edu/software/tophat/manual.shtml
The alignments in the BAM files were further analyzed using the Cufflinks suite of programs (running consecutively: Cufflinks à Cuffcompare à Cuffdiff) http://cole-trapnell-lab.github.io/cufflinks/manual/
Cufflinks was run using the rn5-genes as a reference database (gtf file). By doing so, the output shows the known genes with their RNA-Seq metrics (as FPKM = Fragments Per Kilobase of exon model per Million mapped fragments), but novel transcripts are not identified. The 6 (3 x 2 replicates) cufflinks outputs were compared using cuffdiff. All cuffdiff output files can be opened and converted to Excel tables. This was done for the most important table: “gene_exp.diff”: “3514Rutgers_allcomparisons_gene_exp_diff.xlsx”. By default, genes with q-values <0.05 are designated as “significant” (yes in last column), and in this assay with replicates the q- and p-values are now more meaningful.
The gene_exp.diff table contains all 3 possible pair-wise comparisons. To simplify this table, we generated a derived table that only contains the 2 comparisons between the DEX-treated samples and the Control sample, and with corresponding columns arranged next to each other. This file is called “3514Rutgers_DEXvsCONT_gene_exp_diff.xlsx”. Note that in these tables derived from the gene_exp.diff cuffdiff output, it is best to ignore entries with “NOTEST” in column “status”, as well as short genes (e.g. noncoding small RNA genes). Noncoding RNAs are often located inside mRNA genes and the signals are likely derived from the mRNA gene expression and do not reflect the transcription of the noncoding small RNA gene.
A filtered table with the most differential genes is again included. This time the “significant” columns were used to only keep genes that have “yes” for at least 1 time point. 737 genes are on this list.
Selected graphics generated from the cuffdiff output with the R bioconductor package cummeRbund are included. In the Volcano plots, the genes with q-values <0.05 are indicated with red dots. Please consult online documentation for an interpretation of these plots: http://compbio.mit.edu/cummeRbund/manual_2_0.html
Complete tophat, cufflinks, cuffcompare and cuffdiff outputs are provided for completeness. Please consult the manuals in the links above to find descriptions of all output files. Many of the files are not of immediate interest.
Genome_build: rn5
|
| |
|
| Submission date |
May 22, 2018 |
| Last update date |
Aug 20, 2019 |
| Contact name |
Danish Sayed |
| E-mail(s) |
sayeddh@njms.rutgers.edu
|
| Phone |
(973) 972-5243
|
| Organization name |
Rutgers-NJMS
|
| Department |
CBMM
|
| Lab |
MSB G 626
|
| Street address |
185 south orange avenue
|
| City |
Newark |
| State/province |
NJ |
| ZIP/Postal code |
07103 |
| Country |
USA |
| |
|
| Platform ID |
GPL20084 |
| Series (2) |
| GSE114766 |
The Signature of Glucocorticoid Receptor -Dependent Hypertrophic Transcriptome in Cardiomyocytes [RNA-Seq] |
| GSE114767 |
The Signature of Glucocorticoid Receptor -Dependent Hypertrophic Transcriptome in Cardiomyocytes |
|
| Relations |
| BioSample |
SAMN09237930 |
| SRA |
SRX4112797 |
