A Universal 6iL/E4 Culture System for Deriving and Maintaining Embryonic Stem Cells Across Mammalian Species [EM-seq]

Here, we report the development of a defined, serum-free culture system, termed 6iL/E4, that enables the derivation and long-term self-renewal of ESCs across diverse mammalian species. To further characterize the state of 6iL-hiPSCs, we performed whole-genome bisulfite sequencing (WGBS) to compare 6iL-hiPSCs with hESCs cultured under mTeSR1 and t2iL/Go conditions.

Library PCR amplification was performed for five cycles using NEBNext Multiplex Oligos for Enzymatic Methyl-seq. Equimolar amounts of each library were then pooled and quantified by qPCR before paired-end 150-bp sequencing on a 10B 300-cycle shared flow cell using the NovaSeq X Plus platform at the Advanced Genomics Core.