Total RNA was extracted from human ESCs/iPSCs, d2 PGC-precursor cultures, and d4 and d6 FACS-sorted PGCLC/non–PGCLCs, and mouse PGCLCs and EpiSCs and purified using the RNeasy Micro Kit (QIAGEN, Hilden, Germany). For qPCR, total RNA was reverse-transcribed by M-MLV Reverse Transcriptase (Affymetrix), and the resultant cDNA was used for Q-PCR analysis with iTaq Universal SYBR Green Supermix (Bio-Rad). The primer sequences used are listed in the Supplementary Table S1 .
For transcriptomics microarrays, total RNA was used as input into a linear amplification protocol (Ambion), which involved synthesis of T7-linked double-stranded cDNA and 12 h of in vitro transcription incorporating biotin-labeled nucleotides. Purified and labeled cRNA was then hybridized for 18 h onto HumanHT-12 v4 expression BeadChips (Illumina) for human samples and on MouseRef-8 v2 for mouse samples following the manufacturer's instructions. After washing as recommended, chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and accompanying software. Samples were exclusively hybridized as biological replicates.
For transcriptomics microarrays, total RNA was used as input into a linear amplification protocol (Ambion), which involved synthesis of T7-linked double-stranded cDNA and 12 h of in vitro transcription incorporating biotin-labeled nucleotides. Purified and labeled cRNA was then hybridized for 18 h onto HumanHT-12 v4 expression BeadChips (Illumina) for human samples and on MouseRef-8 v2 for mouse samples following the manufacturer's instructions. After washing as recommended, chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and accompanying software. Samples were exclusively hybridized as biological replicates.