Bulk RNA-Seq of human primordial germ cells induced from human induced pluripotent cells

Mammalian germ cells first establish as primordial germ cells (PGCs). There is an increasing demand to investigate the characteristics of human germ cells including PGCs. Recent studies have established the methodologies to derive human germ cells such as PGCs from human induced pluripotent cells (iPSCs). The in vitro-derived PGCs are referred to as PGC-like cells (PGCLCs) and exhibit the characteristics of the PGCs immediately after specification. Here we performed bulk RNA-sequencing (RNA-Seq) of PGCLCs and their parental iPSCs to characterize the expression signatures of human genes and transposable elements in PGCLCs in detail. Overall design: iPSCs (9A13 XY) line used in this study were established in the previous study (Hwang et al. [PMID: 33168808]). iPSCs were culture on a plate coated with Recombinant laminin-511 E8 (BG iMatrix-511 Silk, Peprotech) and were maintained under a feeder-free condition in the StemFit® Basic04 medium (Asinomoto) containing basic FGF (Peprotech) at 37 °C under an atmosphere of 5% CO2 in air. For the passage or the induction of differentiation, the cells were treated with a 1 to 1 mixture of TrypLE Select (Life Technologies) and 0.5 mM EDTA/PBS to enable their dissociation into single cells, and 10 mM ROCK inhibitor (Y-27632; Tocris) was added. PGCLCs were induced from iPSCs via incipient mesoderm-like cells (iMeLCs) as described previously (Sasaki et al. [PMID: 26189426]) and purified using the surface markers EpCAM and INTEGRINa6. Total RNA was extracted from iPSCs, and PGCLCs by using an RNeasy Micro Kit (QIAGEN) according to the manufacturer's instructions. Synthesis of cDNAs using 1 ng of purified total RNA, and construction of cDNA libraries for RNA sequencing by using an SMART-Seq HT Kit (Takara) and Nextera XT DNA Library Preparation Kit (illumina) according to the manufacturer's instructions. Libraries were sequenced using single-end sequencing protocol on an Illumina NextSeq 500 instrument.