|
| Status |
Public on May 24, 2023 |
| Title |
siBtg2-1 |
| Sample type |
SRA |
| |
|
| Source name |
Neonatal rat ventricular myocyte culture
|
| Organism |
Rattus norvegicus |
| Characteristics |
strain: Sprague-Dawley
tissue: Heart ventricles
genotype: Wildtype
age: Newborn (P0)
treatment: Btg2 siRNA
|
| Treatment protocol |
Transfection cocktails were freshly prepared in Opti-MEM media with Lipofectamine 3000 (Thermo Fisher Scientific, USA). Commercially-available rat siBtg1 and siBtg2, with siScramble as control (Thermo Fisher Scientific, USA) were utilized for single- and double- knockdowns by siRNA. Four treatment groups were as follows: 20nM siScramble (Scramble control), 10nM siBtg1 with 10nM siBtg2 (Btg1/Btg2 double knockdown), 10nM siScramble with 10nM siBtg1 (Btg1 single knockdown), and 10nM siScramble with 10nM siBtg2 (Btg2 single knockdown). Cells were incubated at 37°C for 48 hours.
|
| Growth protocol |
Primary neonatal rat cardiomyocytes were isolated from hearts of newborn rats. Following separation of fibroblasts, cardiomyocyte-enriched media with ~3 million cells were plated on 6-well plates. Cells were grown in 1X Medium-199 with Earle’s salts, L-glutamine, 15% bovine growth serum, and 2X penicillin/streptomycin for ~40 hours to obtain 70 to 80% confluency.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
At 48 hours post-siRNA transfection, cardiomyocyte culture wells were washed in sterile PBS and flash-frozen for RNA isolation. Total RNA was isolated following NucleoSpin RNA Isolation Kit protocol (Macherey-Nagel, Germany).
RNA that passed quality control by the Agilent 2100 Bioanalyzer was amplified using Ovation RNA-Seq System v2. DNA libraries were generated using Nextera XT DNA Library Preparation Kit. Samples were then subjected to paired-end sequencing on Illumina HiSeq 2500 system to a depth of 35-40 million mapped reads.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Sequence reads were trimmed to remove adapter and low quality sequences using FastQC, Trim Galore, and Cutadapt.
Trimmed reads were aligned to the reference rat genome version rn5 with the program STAR.
Aligned reads were stripped of duplicate reads with the program sambamba.
Read counting was done with the program featureCounts from the Rsubread package. Raw counts were normalized as transcripts per million (TPM).
Assembly: rn5
Supplementary files format and content: tab separated file with raw read counts for all samples
Supplementary files format and content: tab separated file with TPM for all samples
|
| |
|
| Submission date |
May 20, 2022 |
| Last update date |
May 24, 2023 |
| Contact name |
Katherine E Yutzey |
| E-mail(s) |
katherine.yutzey@cchmc.org
|
| Phone |
+1 5136365958
|
| Organization name |
Cincinnati Children's Hospital Medical Center
|
| Department |
Molecular Cardiovascular Biology
|
| Lab |
Yutzey
|
| Street address |
3333 Burnet Ave
|
| City |
Cincinnati |
| State/province |
Ohio |
| ZIP/Postal code |
45229 |
| Country |
USA |
| |
|
| Platform ID |
GPL18694 |
| Series (1) |
| GSE203493 |
RNAseq of cultured neonatal rat ventricular cardiomyocytes treated with siRNA for Btg1 and Btg2 knockdown. |
|
| Relations |
| BioSample |
SAMN28571670 |
| SRA |
SRX15403006 |
