NJU-seq of N2A: repeat 1
RNA was fragmented by RNA fragmentation reagents kit (ThermoFisher) at 95 C for 10 mins and then purified by 10 l 3M NaOAc (pH 5.2), 24 l 5 g/l glycogen, and 372 l 96% cold ethanol. After resoluble in water, every 1 g RNA solution was ligated with 3 l 25nM ssRNA adaptor (pAGCUAAAAAAAAAAp, synthesized by GenScript Biotech Co.) at 16 C overnight. The products were then reacted with 30 g MgR in 60 l final volume with 20 mM Tris-Cl (pH 8.5), 100 mM KCl, and 0.01 mM ZnCl2 37 C for 30mins. The products were purified by RNA Clean & Concentration-5 kit (Zymo Research), followed by 3 end 5 phosphorylation (39 l RNA, 5 l (10x T4 PNK Reaction Buffer), 5 l ATP (10 mM), 1 l T4 PNK (3 phosphatase minus, New England BioLabs). We constructed the NGS library using the products from the last step following the NEBNext Small RNA Library Prep Set for Illumina (New England BioLabs) protocol. Fragments smaller than 50nt were extracted from 6% PAGE gel and sent for NGS using Illumina X-Ten. The same process prepared the sample of NC (negative control group) without MgR protein.