Small RNA Library Construction with Improved Adapters

Small RNA libraries were constructed from 20 to 50 nt total RNA according to the Zamore laboratory’s open protocol (https://www.dropbox.com/s/r5d7aj3hhyaborq/) with some modifications (Fu et al, 2018 (link)). The 3′ adapter was conjugated with an amino CA linker instead of dCC at the 3′ end (GeneDesign) and adenylated using a 5′ DNA adenylation kit at the 5′ end (NEB). To reduce ligation bias, four random nucleotides were included in the 3′ and 5′ adapters [(5′-rAppNNNNTGGAATTCTCGGGTGCCAAGG/amino CA linker-3′) and (5′-GUUCAGAGUUCUACAGUCCGACGAUCNNNN-3′)] and adapter ligation was performed in the presence of 20% PEG-8000. After 3′ adapter ligation at 16 °C for ≥ 16 h, the RNAs were size-selected by urea PAGE. For RNA extraction from a polyacrylamide gel, a ZR small RNA PAGE Recovery Kit (ZYMO Research) was used. Small RNA libraries were sequenced on a HiSeq 4000 or DNBSEQ-G400 platform.