Feasible barcode primers

Small RNA libraries were constructed using a NEB Next Ultra Small RNA Sample Library Prep Kit for Illumina according to the manufacturer’s instructions. Reverse transcription primer was hybridized after 3′ adaptor ligation of 10 ng RNA per sample, following 5′ adaptor ligation. Twelve cycles of PCR were performed using Illumina feasible barcode primers after first-strand cDNA synthesis. The prepared libraries were resolved on a native 7% polyacrylamide gel. DNA fragments corresponding to 160–180 bp (including 3′ and 5′ adaptors) were recovered in 10 μL of DNase- and RNase-free water.
Libraries were quantified by the Agilent 2100 bioanalyzer using DNA 1000 chips. A total of 36 sequencing libraries were pooled into a single sequencing lane and sequenced using an Illumina HiSeq4000 analyzer (Illumina, USA).The miRNA sequencing data were quantified, and sequences with a length of more than 18 nt were aligned against miRBase (release 21). The miRNA profiling was normalized using reads per million (RPM) mappable miRNA sequences. Analysis of the differentially expressed miRNAs between the two groups was performed using edgeR software. The Gene Expression Omnibus (GEO) database accession number for the miRNA profile data reported in this study is GEO: GSE147517.