A Universal 6iL/E4 Culture System for Deriving and Maintaining Embryonic Stem Cells Across Mammalian Species

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 cellular states, we performed single-cell RNA sequencing (scRNA-seq) on 6iL-hiPSCs and 6iL-mESCs.

Library preparation was carried out using the Single Cell 3′ RNA Prep T2 kit (Illumina). For indexing, the Mix 7 index set was used for 6iL-hiPSC samples and the Mix 8 index set for 6iL-mESC samples. Sequencing was performed on a NovaSeq X Plus platform using paired-end 150 bp reads (2 × 150 bp), generating approximately 200 million reads per sample (100 million reads per direction).