Genome-wide transcriptomic profiling of cardiomyocyte differentiation from human ES cells and iPS cells under exposure to sublethal of isotretinoin (RNA-seq)

In this study, isotretinoin (INN)-induced alternations in transcriptome during caidiomyocyte differentiation derived from human hESCs and hiPSCs were investigated. H1-hESC and C15-hiPSC were differentiated to caidiomyocytes under exposure to sublethal level of INN, and cells were collected at day 0 (undifferentiated cellsl) day 2 (mesoderm) and day 6 (cardiac progenitors) for genome-wide transcriptomic profiling by RNA-seq. Overall design: H1-hESC and C15-hiPSC were grown in 12-well plates with Essential 8 medium (Thermo Fisher Scientific), and the cardiomyocyte differentiation was initiated using a monolayer differentiation method with PSC Cardiomyocyte Differentiation kit (Thermo Fisher Scientific) under exposure to 25nM of isotretinoin (INN). At day 0, 2 and 6 during the differentiation period (before the medium-change on that day), and cells were collected using Accutase (Thermo Fisher Scientific), and then store in -80C till RNA isolation. For each cell line and each time-point, cells from two independent differentiation wells were used as two biological replicates. RNA-seq libriries were constructed using ScriptSeqâ„¢ v2 RNA-Seq Library Preparation kit (Epicentre Biotechnologies), and then sequenced by a HiSeq 4000 sequencer (Illumina) with 2 X 101 cycles. RNA-seq fastq data were aligned with Tophat (version 2.0.9) to GRCh39/hg19 Homo sapiens reference genome from the UCSC Genome Browser. The human gene symbols and their raw counts were calculated using HTSeq (version 0.6.1p1) package in Python with GRCh39/hg19 Homo sapiens gtf file. Differential gene-expression analysis was performed using edgeR package in R, and the normalization was performed using a trimmed mean of M-values (TMM) method.

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Liu Q, Van Bortle K, Zhang Y, Zhao MT, Zhang JZ, Geller BS, Gruber JJ, Jiang C, Wu JC, Snyder MP