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.