|
| Status |
Public on Nov 04, 2020 |
| Title |
2D_CM_CHIR_3 |
| Sample type |
SRA |
| |
|
| Source name |
human cardiac 2D cultures
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: FACS isolated CD90- (cardiomyocyte) cell populations
|
| Treatment protocol |
human 2D cardiac cultures were treated with either 0.05% DMSO or 5 µM CHIR99021 (Stem Cell Technologies) for 24 hours
|
| Growth protocol |
Briefly, HES3 and H9 hESCs (WiCell) or hiPSC (Sendai-virus reprogrammed CD34+ cells, ATCC) were maintained as TypLE (ThermoFisher Scientific) passaged cultures using mTeSR-1 (Stem Cell Technologies)/Matrigel (Millipore). hPSCs were seeded at 2´104 cells/cm2 in Matrigel-coated flasks and cultured for 4 days using mTeSR-1. They were then differentiated into cardiac mesoderm using RPMI B27- medium (RPMI1640 GlutaMAX+ 2% B27 supplement without insulin, 200 μM L-ascorbic acid 2 phosphate sesquimagnesium salt hydrate (Sigma) and 1% Penicillin/Streptomycin (all ThermoFisher Scientific) containing 5 ng/ml BMP-4 (RnD Systems), 9 ng/ml Activin A (RnD Systems), 5 ng/ml FGF-2 (RnD Systems) and 1 μM CHIR99021 (Stem Cell Technologies) with daily medium exchange for 3 days. Subsequently, they were specified into a cardiomyocyte/stromal cell mixture using RPMI B27- containing 5 μM IWP-4 (Stem Cell Technologies) followed by another 7 days of RPMI B27+ (RPMI1640 GlutaMAX + 2% B27 supplement with insulin, 200 μM L-ascorbic acid 2 phosphate sesquimagnesium salt hydrate and 1% Penicillin/Streptomycin) with medium exchange every 2-3 days. The differentiated cells were then cultured in RPMI B27+ until digestion at 15 days using 0.2% collagenase type I (Sigma) in 20% fetal bovine serum (FBS) in PBS (with Ca2+ and Mg2+) for 60 min at 37°C, followed by 0.25% trypsin-EDTA for 10 min. The cells were filtered using a 100 mm mesh cell strainer (BD Biosciences), centrifuged at 300 x g for 3 min, and resuspended at the required density in CTRL medium: α-MEM GlutaMAX, 10% FBS, 200 μM L-ascorbic acid 2 phosphate sesquimagnesium salt hydrate and 1% Penicillin/Streptomycin.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using trizol and precipitated using glycogen according to manufacturers instructions. Clean-up and DNAse protocols was performed using Rneasy MinElute Cleanup Kit (QIAGEN)
libraries were constructed with Nugen Ovation RNA-Seq system V2 (for SPIA amplifications and cDNA generation) coupled with the Ovation Ultralow System (NuGEN). Libraries were read with 50bp SR Rapid Mode on a HiSeq1500. Barcode information : SampleULN_i7index _LaneNumber_ReadNumber.fastq
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 1500 |
| |
|
| Description |
20200506_CHIRvsDMSO_CPM
20200506_CHIRvsDMSO_Myo
20200506_CHIRvsDMSO_Count_matrix
|
| Data processing |
FASTQ files were quality trimmed with Trimmomatic
Reads were mapped to hg38 or mm10 using RNA STAR
Count matrix was calculated using Htseq tools
EdgeR was used for all comparisons
Genome_build: GRCh38/hg38 or GRCm38/mm10
|
| |
|
| Submission date |
May 13, 2020 |
| Last update date |
Nov 04, 2020 |
| Contact name |
James Hudson |
| E-mail(s) |
James.Hudson@QIMRBerghofer.edu.au
|
| Organization name |
QIMR Berghofer Medical Research Institute
|
| Lab |
Cardiac Bioengineering Laboratory
|
| Street address |
300 Herston Rd
|
| City |
Brisbane |
| State/province |
QLD |
| ZIP/Postal code |
4006 |
| Country |
Australia |
| |
|
| Platform ID |
GPL18460 |
| Series (1) |
| GSE150521 |
β-catenin drives distinct transcriptional networks in proliferative and non-proliferative cardiomyocytes |
|
| Relations |
| BioSample |
SAMN14916062 |
| SRA |
SRX8339213 |
