RNA-seq of mouse embryonic fibroblasts from Eif2b1[N208Y] mice cultured with or without 2BAct
RNA-seq of mouse embryonic fibroblasts from Eif2b1[N208Y] mice cultured with or without 2BAct
WT and N208YHOM mouse embryo fibroblasts (MEFs) were isolated from embryos at embryonic day 13.5. Each embryo was individually processed by first removing the liver and heart before mincing into small pieces followed with trypsin digestion. Cells were mechanically dissociated by pipetting before inhibition the trypsin reaction with complete medium (Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Sigma) and 1X antibiotic-antimycotic solution (Gibco) and 200nM 2BAct).  Cells were cultured in a 15 cm petri dish housed in an incubator at 37°C, 5% CO2, 3% O2 and passaged every 2-3 days with trypsin MEFs were seeded in DMEM with 10% FBS with 200 nM 2BAct. Cells were washed 3 times in PBS prior to replacing cells with media containing with or without 200 nM 2BAct for 6 hours.  Cells were collected in RNA lysis buffer (Applied Biosystems A27828) and RNA was extracted following the manufacturer's protocol for bulk RNA sequencing using MaxMAX total RNA isolation kit (Invitrogen #AM1830) RNA-seq libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA)