Nebnext small rna library prep set for kit

We used the NEBNext Small RNA Library Prep Set for Illumina kit (New England Biolabs) to process and build a small RNA library, according to the manufacturer’s instructions [35 (link)]. We used the Illumina Hiseq2500 for single-terminal sequencing. We processed data and used FastQC (http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/version0.10.1) to detect the original sequence. We compared our BLAST results with the RFam database to annotate the miRNA sequence. Clean reads were compared with human precursor/mature miRNA in miRBase (http://www.mirbase.org/version20.0) to screen for known miRNAs. We compared the uncommented sequence with the human genome to analyze its expression distribution. We used Mireap (http://mireap.sourceforge.net/version0.2) to predict potential new miRNAs. The number of sequences of each known miRNA was standardized according to the total number of sequences paired into the miRBase 20.0 database. The number of sequences per million paired sequences (reads per million mapped reads, RPM) was used as the expression amount. FDR correction was applied to p value to obtain Q value as follows: The screening criteria for differential miRNA were as follows: | log2 (fold change) |≥ 1, P value < 0.05.

Total RNA was extracted using the mirVana miRNA Isolation Kit (Ambion). The RNA quantity was assessed with Nanodrop 2000 (Thermo Fisher Scientific Inc., USA), while its integrity was evaluated using the Agilent 2100 Bioanalyzer (Agilent Technology, USA). To construct small RNA libraries, the NEBNext Small RNA Library Prep Set for Illumina kit (NEB, USA) was utilized. After confirming the high-quality libraries with the Agilent 2100 Bioanalyzer, sequencing was performed on the Illumina Novaseq 6000 platform. For the analysis of differentially expressed miRNAs, the criteria used for filtering were a q-value < 0.05 and fold change (FC) > 2 or FC < 0.5. The DEG algorithm from the R package was employed to calculate q-values. Target gene prediction was carried out using the miranda software, with the parameters set as follows: S ≥ 150, ΔG ≤ -30 kcal/mol, and strict demand for 5’ seed pairing. Finally, differential expression miRNAs’ target genes were subjected to GO enrichment and KEGG pathway enrichment analyses using R packages. All small RNA sequencing and data analyses were performed by Eurofins Genomics (Shanghai, China).

Total RNA was extracted by the mirVana miRNA Isolation Kit (Ambion) following the protocol of instruction. The quantitation of total RNA and evaluation of RNA integrity were conducted as previously described. Total RNA (1000 ng) extracted from each sample was utilized to construct the small RNA libraries with NEBNext Small RNA Library Prep Set for Illumina kit (NEB, USA). In brief, the adapter-ligated RNA underwent reverse transcription to generate cDNA and subsequent amplification with PCR. The products of PCR (140–160 base pairs in length) were isolated to create the miRNA libraries. Next, the libraries were assessed and performed sequencing with the Illumina Novaseq 6000 platform.