GSM5288497: WT_SCNT_E14p5_placenta_RNA_rep2; Mus musculus; RNA-Seq
Spongiotrophoblast from placenta was dissected and lysed with TRIzol reagent (Ambion, Cat. 15596026). RNA was extracted by isopropanol precipitation. 10 µg RNA was DNase I (Fermentas, Cat. EN0521) treated at 37°C for 1 hour. RNA with polyA tails were captured and purified using DynabeadsTM mRNA Purification Kit (ThermoFisher Scientific, 61006). Purified RNA was fragmented with RNA Frag. Buffer (NEB, Cat. E6186A) at 95°C for 5 min. Reaction was then stopped and RNA was purified. First stand cDNA was synthesized with a commercial kit using both oligo dT and random primers (Invitrogen, Cat. 18080-051). Second strand cDNA was synthesized with second strand synthesis buffer (Invitrogen, Cat. 10812014) containing MgCl2, DTT, dNTP, dUTP, RNase H (Fermentas, Cat. EN0202), E. coli DNA ligase (NEB, Cat. M0205S) and DNA polymerase I (NEB, Cat. M0209S). DNA was purified after 2-hour incubation on thermomixer at 16°C. Synthesized cDNA was subjected to library preparation using NEBNext UltraTM II DNA library Prep Kit for Illumina (NEB, E7103) according to manufacturer's instructions.