|
| Status |
Public on Jul 27, 2020 |
| Title |
in vitro12w-2 [12] |
| Sample type |
SRA |
| |
|
| Source name |
human embryonic cell-derived cardiomyocyte
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: H9
tissue/cell type: Cardiomyocytes
developmental/differentiation stage: differentiation day 104
|
| Treatment protocol |
2 x 10^7 human embryonic stem cell-derived cardiomyocytes were directly injected to rat hearts (in vivo samples)
|
| Growth protocol |
Undifferentiated human embryonic stem cells reached 90% confluency, E8 medium was supplied with 1 uM of Wnt activator CHIR99201 (Sigma-Aldrich). The next day (day 0), E8 medium was changed to the cardiac differentiation medium (RPMI 1640 with B27 supplement minus insulin (Gibco) plus L-glutamine adding activin A (100 ng/mL, R&D) and Matrigel. On day1, bone morphologic protein 4 (BMP4; 10 ng/mL, R&D) and CHIR99201, followed by addition of Wnt inhibitor XAV939 (1 uM Sigma-Aldrich) on day 3-4. After day 7, medium was changed to RPMI 1640 with B27 supplement (Gibco) every other day. The cells were heat-shocked at 43ºC for 30 minutes and cryopreserved on day 20.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For collection of in vitro samples, total RNA was extracted by ISOGEN (Nippon Gene), phenol and chloroform. For in vivo samples, rat hearts of each time points were collected and immediately embedded in an OCT-embedding compound and kept in -80℃. Tissues were sectioned at a thickness of 10 µm using a cryostat (Leica). The graft areas were captured using laser microdissection system (Leica) from unstained unfixed specimens attached on the membrane coated slides (Leica 11600289). Total RNA was extracted by Arcturus PicoPure RNA Isolation Kit (Thermo).
cDNA was synthesized by SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (TaKaRa). Library preparations were conducted using Nextera DNA Library Prep Kit
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
HCYYWDSXX_PR1095_12A2206_H1_L004
|
| Data processing |
Sequenced reads were trimmed by Trimmomatic (v0.39) with parameters of SLIDINGWINDOW:10:30. All samples were separated human and rat reads using Xenome (v1.0.0). Reads classified as human were mapped to the hg38 reference with STAR (v2.7.2a) and gene count matrix was generated with featureCounts (v1.6.4).
Differential expression analysis was performed using the edgeR package, with trimmed mean of M values (TMM) normalization. PCA plots were performed using PCAtools package, with transcripts per million (TPM) normalization.
Genome_build: hg38
Supplementary_files_format_and_content: featurecounts_3-12-23-28Enid2 contains raw counts and tpm-normalized_counts.csv contains TPM normalized data.
|
| |
|
| Submission date |
Sep 11, 2019 |
| Last update date |
Jul 27, 2020 |
| Contact name |
Shin Kadota |
| E-mail(s) |
shinkadota@shinshu-u.ac.jp
|
| Organization name |
Shinshu University
|
| Department |
Regenerative Science and Medicine
|
| Street address |
3-1-1 Asahi
|
| City |
Matsumoto |
| ZIP/Postal code |
390-8621 |
| Country |
Japan |
| |
|
| Platform ID |
GPL24676 |
| Series (1) |
| GSE137255 |
Gene expression profilings in human embryonic stem cell-derived cardiomyocytes in vivo and in vitro |
|
| Relations |
| BioSample |
SAMN12727030 |
| SRA |
SRX6830262 |
