GSM5584029: Epilepsy_rep2_control; Mus musculus; RNA-Seq
Total RNA was isolated from cell lines or mouse tissues with TRIzol (Invitrogen) according to the manufacture's protocol. The mRNA was isolated by subjecting total RNA to oligo(dT) enrichment using Dynabeads Oligo(dT)25(NEB) and gDNA was removed by TURBO DNase (Invitrogen). Purified polyadenylated RNA was fragmented using RNA fragmentation reagents (Ambion) at 70 °C for 10 min. Fragmented RNA was incubated with freshly prepared 50 mM NaIO4 at 25°C for 30 min in the dark.The RNA cleave assays were performed using Endonuclease V (NEB) with standard reaction buffer (10mM Tris-HCl pH 7.5, 0.5mM MnCl2, 50mM KCl, 1mM dithiothreitol and 5% glycerol)  and then perform next-generation sequencing (NGS). Fragmented RNA was first subjected to end repair by T4 PNK. For the treated sample, the RNA was treated with periodate followed by Endonuclease V cleavage.3′-adapter ligation (3′ adapter, /5rApp/NNNNNNTGGAATTCTCGGGTGCCAAGG/3ddC/) followed by the 3′ adapter removing by 5′-deadenylase and RecJf digestion. After purification, SuperScript III was used to perform RT (RT primer, /ACACGACGCTCTTCCGATCT). After cDNA synthesis, 5′ adapter (5′ adapter, /5Phos/NNNNNNNNNNAGATCGGAAGAGCACACGTCTG/3ddC/) ligation were performed. After purification, qPCR was performed to evaluate the cycle numbers of each samples to avoid over-amplification. Library PCR amplification was performed using the NEB primers. The products were purified by NEBNext Sample Purification Beads or low melting point agarose gel and used for sequencing.