Induced pluripotent stem cells of patients with Tetralogy of Fallot reveal transcriptional alterations in cardiomyocyte differentiation

We generated induced pluripotent stem cells (iPSCs) of two patients with Tetralogy of Fallot (TOF) and three healthy relatives of two unrelated familes. We furhter performed mRNA sequencing of iPSCs (day 0) and derived cardiomyocytes (CMs) at day 15 and 60 of patients and healthy relatives. Overall design: Total RNA was isolated using Direct-zol RNA Kits (Zymo Reasearch) and purified with Dynabeads Oligo(dT) (Thermo Fisher Scientific) to get poly(A) RNA. The RNA libraries were prepared with the ScriptSeq v2 RNA-Seq Library Preparation Kit (Illumina). The libraries were evaluated with the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and the Agilent High Sensitivity DNA Kit (Agilent Technologies). Paired-end sequencing was performed on either HiSeq 2500 (2x51 bp) or NextSeq (2x76 bp) device (Illumina). For the latter, the NextSeq 500/550 v2 kits (Illumina) were used. All kits were applied according to the manufacturer's instructions. Overall, mRNA sequencing was performed for 31 samples of 5 individuals for the following three differentiation stages: pluripotent iPSC (day 0), iPSC-CM (day 15) and CM (day 60). Sequencing was separated into two runs, all day 0 samples and three day 15 samples (Mother TOF-02) were sequenced with Illumina's HiSeq 2500. The remaining samples were sequenced using Illumina's NextSeq.

本研究获取了2例法洛四联症（Tetralogy of Fallot, TOF）患者的诱导多能干细胞（induced pluripotent stem cells, iPSCs），以及2个无关家系中3名健康亲属的诱导多能干细胞。随后，我们对患者及健康亲属的诱导多能干细胞（第0天）以及分化至第15天、第60天的心肌细胞（cardiomyocytes, CMs）进行了mRNA测序。
实验设计概况：总RNA采用Direct-zol RNA提取试剂盒（Zymo Research）进行分离，并通过Dynabeads Oligo(dT)磁珠（Thermo Fisher Scientific）纯化以获取聚腺苷酸（poly(A)）RNA。RNA文库构建采用ScriptSeq v2 RNA测序文库制备试剂盒（Illumina）完成。文库质量通过Qubit dsDNA HS检测试剂盒（Thermo Fisher Scientific）与Agilent高灵敏度DNA检测试剂盒（Agilent Technologies）进行评估。随后采用Illumina的HiSeq 2500平台（双端测序，读长2×51 bp）或NextSeq平台（双端测序，读长2×76 bp）进行双端测序；针对后者测序平台，使用Illumina NextSeq 500/550 v2测序试剂盒完成测序。所有实验操作均严格按照试剂盒厂商说明书进行。
综上，本研究针对5名受试者的31份样本，开展了三个分化阶段的mRNA测序，分别为多能态诱导多能干细胞（第0天）、诱导多能干细胞分化心肌细胞（第15天）以及成熟心肌细胞（第60天）。测序分为两轮进行：所有第0天样本与3份第15天样本（TOF-02号受试者母亲）通过Illumina HiSeq 2500平台完成测序，剩余样本则采用Illumina NextSeq平台进行测序。