GSM2052298: mESC_Tet1CS_RNA RNA-seq; Mus musculus; RNA-Seq
Cells were lysed using TRIzol reagent (Ambion, Cat. 15596026). RNA was extracted by isopropanol precipitation and diluted in DEPC-treated water. 10ug RNA was DNase I (Fermentas, Cat. EN0521) treated at 37°C for 1 hour. RNA was purified using AMPure beads. Then ribosomal RNA was removed using Ribo-Zero Magnetic Gold Kit (Epicentre). Purified RNA was fragmented with RNA Frag. Buffer (NEB, Cat. E6186A) at 95°C for 5 min. Reaction was stopped and RNA was purified. First stand cDNA was synthesized with 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), MgCl2, DTT, dUNTP, 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. Then, synthesized cDNA was end-repaired, adenylated and ligated to TruSeq sequencing adaptors. Libraries were then treated with UDG (AMPErase, Cat. N808-0096) at 37°C for 1 hour and purified before PCR amplification. 200-500bp fragments were selected for subsequent sequencing.
GEO Accession
GSM2052298