Nebnext multiplex small rna library

Total RNAs of exosomes were obtained using MiniBEST Universal RNA Extraction Kit (Takara, Japan). The concentration and quality of total RNA was determined by NanoDrop ND1000 (Thermo Fisher Scientific, United States). Reverse transcription reaction and gene library preparation were performed using NEBNext^® Multiplex Small RNA Library (E7300L, NEB, United States) according to the manufacturer’s instructions, followed by assessment of the final library product using the Agilent Bioanalyzer 2100 system (Agilent, United States). The library was then sequenced in the Illumina HiSeq 4000 platform using the 150 bp paired-end sequencing strategy. Cluster 3.0 software (United States) was used to generate a heat map of DE miRNA between the two groups (FDR ≤ 0.001 and | Log2Ratio| ≥ 1). Gene ontology (GO) enrichment was analyzed using Gene Ontology Enrichment Analysis Software Toolkit (GOEAST) with default parameters, including the molecular functions, biological processes, and cellular components. KEGG Orthology Based Annotation System (KOBAS) software was used to analyze the Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathways for the differential expressions of miRNAs.

After the extracted samples were qualified, the Illumina's NEB Next Multiplex Small RNA Library (NEB, USA) was used to prepare the centralized small RNA libraries. Using the unique structures at the 3′ and 5′ ends of the small RNA, total RNA was used as the starting sample, and both ends of the small RNA were directly added with adapters with cDNA synthesized by reverse transcription. Subsequently, after qPCR amplification, DNA fragments of length 140-160 bp were separated by polyacrylamide gel (PAGE) electrophoresis with the cDNA library recovered by cutting the gel. The Qubit 2.0 was then used for preliminary quantification. The effective concentration of the library was accurately quantified by the qPCR method to ensure library quality. The library quality was evaluated using the Agilent Bioanalyzer 2100 system (Agilent Company, USA) and a DNA high-sensitivity chips.