NextSeq 500 sequencing; RNA-seq of murine ES cells with Lima1 knockout, murine EpiS cells with Lima1 overexpression and human iPS cells with LIMA1 overexpression
Cells were collected from culture using standard procedures. Mouse epiblast stem cells were cultured in conditioned medium (CF-1 feeders) with 5 ng/ml FGF2 and 10 ng/ml Activin A. Total RNA was isolated using Macherey-Nagel RNA isolation kit. 1ug of this was used for library preparation and sequencing. Enrichment of mRNA was performed using NEBNext® Poly(A) mRNA Magnetic Isolation Module and cDNA library was constructed using NEBNext® Ultra™ II Directional RNA Library Prep Kit.