hESCs, PreME, ME, DE, hPGCLCs and hPGCs were sorted directly into extraction buffer of PicoPure RNA Isolation Kit (Applied Biosystems) and RNA was extracted according to manufacturer’s protocol with on-column DNase I treatment (Qiagen 79254). RNA-seq libraries were generated from 5 ng total RNA using Ovation RNA-Seq System V2 (Nugen) and Ovation Rapid DR Multiplex System (Nugen)25 (link). Libraries were quantified by qPCR using KAPA Library Quantification Kit (Kapa Biosystems) using QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems) and validated using Agilent TapeStation 2200 with High Sensitivity D1000 ScreenTape. Libraries were subjected to single-end 50 bp sequencing on HiSeq 4000 sequencing system (Illumina), resulting in >30 millions single-end reads per sample.
RNA-seq libraries of PreME aggregate with SOX17 or PRDM1 overexpression were generated by the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB, E7760S) and the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, E7490) according to manufacturer’s protocol. Quantified and validated libraries were subjected to single-end sequencing on HiSeq 4000 sequencing system (Illumina).
RNA-seq libraries of PreME aggregate with SOX17 or PRDM1 overexpression were generated by the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB, E7760S) and the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, E7490) according to manufacturer’s protocol. Quantified and validated libraries were subjected to single-end sequencing on HiSeq 4000 sequencing system (Illumina).
