Agilent bioanalyzer 2100 system s rna nano 6000 assay kit

We used the miRNeasy Mini kit (Qiagen, cat. No. 217004) to extract and purify total RNA from exosomes following the manufacturer’s instructions. Using the Agilent Bioanalyzer 2100 System’s RNA Nano 6000 Assay Kit, we assessed RNA concentration and purity (Agilent Technologies, CA, USA). According to the instructions of the manufacturer, we created sequencing libraries using the QIAseq miRNA Library Kit (Qiagen, Frederick, MD) and added index codes to assign sequences to each of the three samples. The library preparations were sequenced on an Illumina NovaSeq 6000 platform, with paired-end reads produced. After controlling the raw data and filtering repeats and ncRNAs (ribosomal RNA, transfer RNA, small nuclear RNA, and small nucleolar RNA), we aligned the sequencing reads to the miRbase and Human Genome (GRCh38) to identify recognized miRNAs. The transcript per million (TPM) value was obtained from the expression matrix of quantified miRNA UMI counts, and differential analysis was carried out using the EdgeR package. The consequences and sophisticated functioning of biological systems were investigated via the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (www.genome.jp/kegg/). We tested the statistical enrichment of coexpressed genes in KEGG pathways utilizing an R script, and we calculated the p value using Fisher’s exact test.

The Illumina se50 platform was used to sequence RNA from M1-Exos and M2-Exos (NEB, United States). The Agilent Bioanalyzer 2,100 system’s RNA Nano 6000 Assay Kit (Agilent Technologies, CA, United States) was utilized to test RNA integrity. Multiplex Small RNA Library Prep Set for Illumina (NEB, United States) was employed for building sequencing libraries, and index codes were added to each sample’s sequences. DNA High Sensitivity Chips were used to evaluate library efficiency on the Agilent Bioanalyzer 2,100 device. Small RNAs were reverse transcribed, amplified, and sequenced on the Illumina HiSeq 2,500 platform, which provided 50-bp single-end reads. The samples were analyzed by DESeq based on negative binomial distribution and evaluated by fold change and Qvalue. The differential microRNAs were screened with Qvalue < 0.05 and log2 (foldchange) > 1. The overall distribution of differentially represented miRNAs is inferred using a volcanic map.