Small RNA Sequencing and Analysis

Library construction, sequencing, and data analysis were entrusted to Novogene (Tianjin, China). Sequencing libraries were generated using NEBNext Multiplex according to the manufacturer’s recommendations. The small RNA (sRNA) molecules were ligated to a 5′ adaptor and a 3′ adaptor using T4 RNA ligase 1. Then, first-strand cDNA was synthesized using M-MuLV reverse transcriptase. PCR amplification was performed using LongAmp Taq 2× master mix (NEB, USA). At last, library quality was assessed on the Agilent Bioanalyzer 2100 system using DNA High-Sensitivity Chips (Agilent Technologies, USA). After cluster generation, the library preparations were sequenced on an Illumina HiSeq 2500/2000 platform, and 50-bp single-end reads were generated.
Raw reads were first processed through custom perl and python scripts. In this step, clean reads were obtained by removing low-quality reads. The small RNA tags were mapped to reference sequence by Bowtie without mismatch to analyze their expression and distribution on the reference. Using miRBase (http://www.mirbase.org) as reference, modified software miRDeep2 was used to obtain the potential miRNA, and miREvo and miRDeep2 were integrated to predict novel miRNA (79 (link), 80 (link)). Expression analysis of miRNAs was performed using the same method as used for circRNAs.