|
| Status |
Public on Jul 30, 2018 |
| Title |
Sham_1_19 |
| Sample type |
SRA |
| |
|
| Source name |
Cardiomyocytes
|
| Organism |
Mus musculus |
| Characteristics |
operation: Sham
single cell or bulk: single cell
|
| Treatment protocol |
Mice (8 weeks old) underwent transverse aortic constriction (TAC) to induce heart failure or a sham operation. The transverse aorta was constricted with a 7-0 silk suture paralleled with a 27-gauge blunt needle, which was removed after constriction. Sham-operated mice, which were used as controls, were consisted of mice that had undergone a similar surgical procedure without aortic constriction 2 weeks previously. Transthoracic echocardiography was performed on conscious mice with a Vevo 2100 imaging system (VisualSonics). M-mode echocardiographic images were obtained from a longitudinal view to measure left ventricular size and function.
|
| Growth protocol |
C57BL/6 mice were purchased from CLEA JAPAN and α-MHC-Cretg mice were from Jackson Laboratory. Trp53flox/flox mice were obtained from Dr. Anton Berns (The Netherlands Cancer Institute, Amsterdam, Netherlands). All mice were maintained in specific pathogen-free conditions in the animal facilities of the University of Tokyo and Osaka University. All animal experiments were approved by Ethics Committee for Animal Experiments of the University of Tokyo and Osaka University and strictly adhered to the animal experiment guidelines. Conditional deletion of Trp53 in cardiomyocytes was achieved by crossing Trp53flox/flox homozygous mice with α-MHC-Cretg hemizygous mice. Trp53flox/flox; α-MHC-Cretg (Trp53α-MHC-Cre) mice were used as p53 cardiomyocyte-specific knockout (p53CKO) mice. Male mice at 8 weeks of age were used for all experiments. All mice were on a C58BL/6 background.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Cardiomyocytes were isolated using Langendorff perfusion from the left ventricular free wall 2 weeks after the sham operation and 3 days and 1, 2, 4 and 8 weeks after the TAC operation. Echocardiography was used to evaluate left ventricular function and assess whether the heart was appropriately exposed to pressure overload. Rod-shaped live cardiomyocytes (viability of cardiomyocytes was over 80% for all time points) were immediately collected from two mice at each time point with a 0.2–2-µl pipette (sample volume, 0.5 µl) and incubated in lysis buffer.
Single-cell cDNA libraries were generated using the Smart-seq2 protocol and the efficiency of reverse transcription was assessed by examining the Ct (Cycle threshold) values from real-time quantitative RT-PCR of control genes (Tnnt2, Cox6a2) using a CFX96 real-time PCR detection system (Bio-Rad) and by examining the distribution of the lengths of cDNA fragments using a LabChip GX (Perkin Elmer) and/or TapeStation 2200 (Agilent Technologies). The following primer sets were used for real-time quantitative RT-PCR: Tnnt2 mRNA forward, TCCTGGCAGA GAGGAGGAAG; Tnnt2 mRNA reverse, TGCAGGTCGA ACTTCTCAGC; Cox6a2 mRNA forward, CGTAGCCCTC TGCTCCCTTA; Cox6a2 mRNA reverse, GGATGCGGAG GTGGTGATAC. A Ct value of 25 was set as threshold. The remaining libraries were sequenced using a Hiseq 2500 system (Illumina).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
single-cardiomyocyte RNA-seq.txt
|
| Data processing |
Reads were mapped to the mouse genome (mm9) using Bowtie (version 1.1.1) with the parameters ‘-S -m 1 -l 36 -n 2 mm9’.
RPKM was calculated with reads mapped to the nuclear genome using DEGseq (version 1.8.0).
Genome_build: mm9
Supplementary_files_format_and_content: Tab-delimited text files including RPKM values for each sample
|
| |
|
| Submission date |
Feb 21, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Seitaro Nomura |
| E-mail(s) |
senomura-cib@umin.ac.jp
|
| Phone |
81338155411
|
| Organization name |
The University of Tokyo
|
| Department |
Department of Cardiovascular Medicine
|
| Street address |
7-3-1, Hongo, Bunkyo-ku
|
| City |
Tokyo |
| State/province |
Select a State or Province |
| ZIP/Postal code |
113-8655 |
| Country |
Japan |
| |
|
| Platform ID |
GPL17021 |
| Series (2) |
| GSE95140 |
Cardiomyocyte gene programs encoding morphological and functional signatures in cardiac hypertrophy and failure (RNA-Seq) |
| GSE95143 |
Cardiomyocyte stress-response gene modules regulate cardiac hypertrophy and failure |
|
| Relations |
| BioSample |
SAMN06352674 |
| SRA |
SRX2580316 |
