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 embryonic stem cells were maintained in N2B27 in the presence of 2i/Lif (0.4 µM PD0325901, 3 µM CHIR99021 and 4 ng/ml Lif), on plastic dishes coated with FCS. 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.