miRNA Sequencing Library Prep and Analysis

NEB Next Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs) was performed to construct miRNA sequencing libraries. The quality of the libraries was evaluated by a Bioanalyser 2100 system (Agilent Technologies). Then, the single-stranded DNA molecules from denatured libraries were captured on Illumina flow cells and amplified in situ as clusters. A HiSeq 4000 sequencing system (Illumina) was used for sequencing for 50 cycles according to the manufacturer’s instructions. Cutadapt software (v1.9.3) was used to trim the adaptor sequences from the sequencing data and retain the adaptor-trimmed reads (> = 15 nt). The trimmed reads were merged and used to predict novel miRNAs with miRDeep2 software (v2.0.0.5) [22 (link)]. The alignment of the trimmed reads was used both miRBase (http://www.mirbase.org) to obtain known pre-miRNAs and Novoalign software (v3.02.12) (http://www.novocraft.com/main/index.php) for newly predicted pre-miRNAs. The primal expression levels of the miRNA were determined via the numbers of tags on each mature miRNA. The TPM (tag counts per million aligned miRNAs) method was used for the standardization of read counts.