|
| Status |
Public on Dec 13, 2016 |
| Title |
Adult rat cardiomyocyte [adult_H] |
| Sample type |
SRA |
| |
|
| Source name |
isolated from ventricular heart tissue
|
| Organism |
Rattus norvegicus |
| Characteristics |
genotype/variation: WT
age: adult
|
| Growth protocol |
ES cells were differentiated into cardiomyocytes and either transplanted into a neonatal rat heart or cultured in vitro for 30 days
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Single cardiomyocytes were FACS sorted (SH800, Sony Technologies) or manually picked under a microscope.
Cells were placed into 96 well plates in water DNase I and RNase inhibitor. DNase was inactivated by increasing the temperature to 72 degrees Celsius for 3 minutes and then cells were placed on ice. SMARTscribe reverse transcriptase, RNase inhibitor, DTT, dNTP, MgCl2, and custom primers were used to convert the mRNA to cDNA. A custom designed PCR primer was used with Advantage2 taq polymerase and was added to the reverse transcription product and dNTP. This was amplified for 19 cycles and was purified using Ampure XP beads (Beckman-Coulter). 100 pg of total cDNA was added to a tagment DNA buffer and spiked with Tn5 (Epicenter) and incubated for 8 minutes at 55 degrees C, which was then stripped off by adding 0.2% SDS to a concentration of 0.05%. Libraries were enriched using KAPAHiFi with index primers and then multiplexed and sequenced.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
adult_H
|
| Data processing |
Reads were aligned to the mouse reference genome using Tophat (2.1.0)
Aligned reads were assembled using Htseq-count
Differential expression analysis was performed using SCDE package and DEseq2 in R
Gene ontology analysis results were visualized using Revigo
Genome_build: mm10
Supplementary_files_format_and_content: rawcounts.csv contains the output of Htseq-count package with all genes with at least one read for 24 single cells. Each column represents and individual cell and the rows have the gene names.
|
| |
|
| Submission date |
Dec 12, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Chulan Kwon |
| E-mail(s) |
ckwon13@jhmi.edu
|
| Organization name |
Johns Hopkins University
|
| Department |
Medicine, Cardiology, Heart & Vascular Institute, Institute for Cell Engineering, and Cellular and Molecular Medicine
|
| Street address |
720 Rutland Ave, Ross 954
|
| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21042 |
| Country |
USA |
| |
|
| Platform ID |
GPL20084 |
| Series (1) |
| GSE92247 |
Neonatal Transplantation Confers Maturation of PSC-Derived Cardiomyocytes Conducive to Modeling Cardiomyopathy |
|
| Relations |
| BioSample |
SAMN06131464 |
| SRA |
SRX2414765 |
