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. 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 ScriptSeq™ 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.

本研究旨在探究异维A酸（isotretinoin，INN）对人胚胎干细胞（human embryonic stem cells, hESCs）与诱导多能干细胞（human induced pluripotent stem cells, hiPSCs）分化为心肌细胞过程中转录组的调控变化。实验中，将H1-hESC细胞系与C15-hiPSC细胞系置于含亚致死浓度异维A酸的培养体系中诱导分化为心肌细胞，并分别在分化第0天（未分化细胞）、第2天（中胚层细胞）及第6天（心肌祖细胞）收集细胞，通过RNA测序（RNA-seq）开展全基因组转录组分析。细胞均接种于12孔板，采用Essential 8培养基（Thermo Fisher Scientific）培养；使用PSC心肌细胞分化试剂盒（Thermo Fisher Scientific），通过单层细胞分化法启动心肌细胞分化，体系中添加25纳摩尔浓度的异维A酸（INN）。分别于分化第0、2、6天（当日换液前）收集细胞，采用Accutase细胞消化液（Thermo Fisher Scientific）解离后，将细胞冻存于-80℃冰箱直至RNA提取。针对每一株细胞系与每一个时间点，均取两个独立分化孔的细胞作为生物学重复（biological replicates）。采用ScriptSeq™ v2 RNA测序文库制备试剂盒（Epicentre Biotechnologies）构建RNA测序文库，随后通过Illumina HiSeq 4000测序仪以2×101个循环的模式完成测序。将RNA测序得到的fastq格式数据，通过Tophat软件（版本2.0.9）比对至UCSC基因组浏览器提供的GRCh39/hg19人类参考基因组。使用Python环境下的HTSeq软件包（版本0.6.1p1），结合GRCh39/hg19人类基因组GTF注释文件（GTF file），计算得到人类基因符号及其原始计数。采用R语言中的edgeR软件包开展差异基因表达分析，并通过截尾均值归一化（trimmed mean of M-values, TMM）法完成数据标准化。