Tyrosine Kinase Inhibitor-induced cardiotoxicity in hiPSC derived cardiomyocytes - pilot dataset - Dataset (ID:20324)
|HMS Dataset ID:||20324|
|Dataset Title:||Tyrosine Kinase Inhibitor-induced cardiotoxicity in hiPSC derived cardiomyocytes - pilot dataset|
|Screening Lab Investigator:||Huan (Sharon) Wang, Sarah Boswell|
|Screening Principal Investigator:||Peter K. Sorger|
|Assay Description:||To define molecular network markers of tyrosine kinase inhibitor-induced cardiotoxicity, we treated human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with one of four tyrosine kinase inhibitors displaying a range of mild to severe effects on heart function (Erlotinib, Lapatinib, Sorafenib, or Sunitinib) or vehicle control (DMSO). We performed 6 different treatments for each drug, including three doses (1, 3.16 and 10 µM) at 24 hours and the intermediate dose (3.16 µM) for an additional three time points (6h, 72h and 168h). Gene expression changes were assessed at the cell population level using total RNA-Seq, which measured levels of both mRNAs and long non-coding RNAs. Validation of RNA-Seq results was performed by qPCR (HMS-LINCS Dataset #20325).|
1. Cryo-preserved Cor.4U hiPSC-CMs (Ncardia, previously Axiogenesis) were thawed into 75cm2 flasks for two days before reseeding into multi-well plates in order to remove cells that died during the thawing process.|
2. Media was changed every other day during culture.
3. Cor.4U hiPSC-CMs were cultured in BMCC media (Ncardia, previously Axiogenesis) containing 1% fetal bovine serum during drug treatment.
4. A different batch of Cor.4U cells was used in each of three independent drug treatment experiments.
5. Cells were washed once with PBS with no calcium or magnesium, scraped in RLT buffer (RNeasy Mini Kit, Qiagen), and cell lysates were frozen at -80 °C until further processing.
6. At the time of RNA purification, samples were thawed at room temperature and processed with QIAshredder (Qiagen) to ensure complete lysis.
7. Total RNA purification was performed following manufacturer’s instructions, including a 30 minute on-column DNase digestion step. Sample concentrations were determined by Nanodrop and RNA quality was assessed on a subset of samples by Bioanalyzer (Agilent); all samples scored RINs of > 9.0.
8. RNA sequencing library preparation was performed with the High Throughput TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero gold (Illumina) following the manufacturer’s protocol. Input for each sample consisted of 500ng of RNA and 1ul of 1:100 diluted ERCC spike-in mix A (Ambion). Libraries were amplified for 15 cycles during the final amplification step.
9. Libraries were quantified using the Qubit dsDNA HS assay (Thermo Fisher Scientific).
10. Library size and quality were spot checked for a subset of samples by Bioanalyzer (Agilent). The average size of cDNA fragments in the libraries was 330 base pairs.
11. Libraries were pooled at equimolar concentrations and sequenced using HiSeq 2500 v4 (Illumina) at the Bauer Core Facility (Harvard University).
12. We used the STAR algorithm in the bcbio-nextgen suite (bcbio-nextgen.readthedocs.io/en/latest/index.html) to map reads onto human genome build GRCh37. Overall, we obtained 10-15 million mapped reads per sample and most RNA samples had very low level of rRNA reads, except three of the samples (dmso-168-0_001-1.R1.fastq.gz, sorafenib-168-3-2.R1.fastq.gz, sunitinib-24-10-2.R1.fastq.gz and their corresponding pair-ended files) which were removed from subsequent differential expression analyses.
|HMS Dataset Type:||RNA-Seq|
|Date Publicly Available:||2018-03-20|
|Most Recent Update:||2018-03-23|