Small RNA library construction, sequence analysis, and
identification of miRNAs
The experimental procedure mainly includes two parts: preparation of
Library and sequencing experiment. Small RNA sequencing library
preparation using TruSeq Small RNA Sample Prep Kits (Illumina, San
Diego, USA). After library preparation, the libraries were sequenced
using Illumina Hiseq2000/2500 with a single-end 1X50bp read length.
The miRNAs data analysis software is the self-developed ACGT101-miR (LC
Sciences, Houston, Texas, USA). The analysis flow of the software is
shown in Figure 1B. In brief, Remove 3’ connectors and rubbish sequences
to get clean data and screening of sequences with base length between
18-26nt. Then, the remaining sequences were compared to (without miRNA)
mRNA, RFam and Repbase databases and filtered and obtaining valid data
and comparing precursors and genomes for miRNAs identification. The
miRNA identification base is linked to the miRBase and the genome of the
species, and the degree of accuracy is highly correlated with the
completeness of the database. Clean Data was used to identify small RNAs
and calculate the miRNAs expression levels identified in each sample
using ACGT101-miR. Expression was counted and used to assess the
correlation of gene expression characteristics and differentially
expressed miRNAs within and between groups of samples.