title: NextSeq 500 paired end sequencing; RNA-seq of wild-type mouse embryonic stem cells (mESC) during spontaneous differentiation
design: RNA-seq of wild-type mouse embryonic stem cells (mESC) during spontaneous differentiation
library_construction_protocol: Mouse embryonic stem cells (mESC)-R1 (kindly provided by Alexander Aulehla, EMBL) were maintained at 37 C and 5% CO2 without feeder cells in DMEM high glucose (Gibco, 41965) supplemented with 15% ES Cell-qualified EmbryoMax Fetal Bovine Serum (Merck Millipore, ES-009-B), penicillin/streptomycin (Sigma Aldrich, P0781), 2 mM L-glutamine (Sigma-Aldrich, G7513), non-essential amino acids (Gibco, 11140), 1 mM sodium pyruvate (Gibco, 11360), 0.1 mM 2-mercaptoethanol and LIF (EMBL Protein Expression and Purification Core Facility), on culture dishes (Nunc) coated with 0.1% gelatin (Sigma-Aldrich, G1890) solution. Pluripotent mESCs were incubated for 2 days in medium with LIF, or seeded at a concentration of 10.4 cells/mm2 in medium without LIF and incubated for 3 or 7 days exchanging the medium every other day, and processed.  Cell monolayer was washed with PBS and total RNA was purified with TRIreagent (Sigma-Aldrich, 9424) following manufacturer??s instructions.        Barcoded stranded RNAseq libraries were generated from high quality RNA samples (500 ng/sample) with the Illumina TruSeq Stranded mRNA Sample Preparation kit following manufacturerâ€™s indications, using a liquid handling robotic system Beckman FXP2.