title: NextSeq 500 paired end sequencing; RNA-seq of wild-type, Gal-1 knockout, Gal-3 knockout and Gal-1/Gal-3 double knockout mouse embryonic stem cells (mESC) deprived of LIF for 7 days
design: RNA-seq of wild-type, Gal-1 knockout, Gal-3 knockout and Gal-1/Gal-3 double knockout mouse embryonic stem cells (mESC) deprived of LIF for 7 days
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 wild-type, Gal-1 -/-, Gal-3-/- and Gal-1-/-; Gal-3-/- mESCs were seeded at a concentration of 10.4 cells/mm2 on 6-well plates in medium without LIF. Cells were incubated for 7 days in the absence of LIF exchanging the medium every other day and processed. Cell monolayer was washed with PBS and total RNA was extracted with the Quick-RNA Miniprep kit (Zymo Research, R1054) following manufacturer??s instructions. RNA was eluted in 100 microliters of water and concentrated using a SpeedVac until reaching a volume of ~37 microliters, treated with 2 microliters of DNase I (Takara, 2270B) in the presence of RNase inhibitors (EMBL Protein Expression and Purification Core Facility) and re-purified using the Quick-RNA Microprep kit (Zymo Research, R1050). RNA integrity was assessed in a 2100 Bioanalyzer System (Agilent). Barcoded stranded RNAseq libraries were generated from high quality RNA samples (400 ng/sample) with the Illumina TruSeq Stranded mRNA Sample Preparation kit following manufacturerâ€™s indications, using a liquid handling robotic system Beckman FXP2.