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Dataset Information
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Dataset Name:
Tyrosine Kinase Inhibitor-induced cardiotoxicity in hiPSC derived cardiomyocytes - pilot dataset (qPCR validation)

Dataset 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 3 different treatments for each drug, including two doses (3.16 and 10  M) at 24 hours and the lower dose (3.16  M) at 72h. Gene expression changes were assessed at the cell population level using qPCR. This is a validation dataset for RNA-seq HMS-LINCS Dataset <a href = "http://lincs.hms.harvard.edu/db/datasets/20324/">#20324</a>. 

--Data in Package:
20325.txt

--Metadata in Package:
Small_Molecule_Metadata.txt

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Center-specific Information
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Center-specific Name:
HMS_LINCS

Center-specific Dataset ID:
20325

Center-specific Dataset Link:
http://lincs.hms.harvard.edu/db/datasets/20325/

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Assay Information
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Assay Protocol:
1. Cryo-preserved Cor.4U hiPSC-CMs (Ncardia, previously Axiogenesis) were thawed into 75cm<sup>2</sup> flasks for two days before reseeding into multi-well plates in order to remove cells that died during the thawing process. <br /> 
2. Media was changed every other day during culture.<br />
3. Cor.4U hiPSC-CMs were cultured in BMCC media (Ncardia, previously Axiogenesis) containing 1% fetal bovine serum during drug treatment.<br />
4. The same batch of Cor.4U cells was used for each of the three technical replicates of drug treatment conditions in this experiment.<br /> 
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.<br /> 
6. At the time of RNA purification, samples were thawed at room temperature and processed with QIAshredder (Qiagen) to ensure complete lysis.<br /> 
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.<br />
8. Samples were reverse transcribed to cDNA using an RT2 First Strand Kit (Qiagen, Cat. No. 330404).<br /> 
9. Gene expression of 90 selected genes and four housekeeping genes (ACTB, GUSB, NONO, RPLP0) were measured based on custom RT2 profiler PCA arrays using RT SYBR Green ROX qPCR Mastermix (Qiagen, Cat. No. 330524) and Applied Biosystems QuantStudio 7 Flex Real-Time PCR System (ThermoFisher Scientific). Experiments were performed by the Qiagen Lifesciences Service Core (Frederick, MD). .<br />
10. Ct values were exported to a text file and analyzed using R, and normalized to the average Ct values of three of the housekeeping genes (GUSB, NONO, RPLP0) whose expression was determined to be unchanged among the experimental conditions.<br /> 

Date Updated:
2018-03-23

Date Retrieved from Center:
5/21/2018

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Metadata Information
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Metadata information regarding the entities used in the experiments is included in the accompanied metadata. A metadata file per entity category is included in the package. For example, the metadata for all the cell lines that were used in the dataset are included in the Cell_Lines_Metadata.txt file.
Descriptions for each metadata field can be found here: http://www.lincsproject.org/data/data-standards/
[/generic/reagents_studied]
