Hiseq 2500 system

NGS samples were prepared by pooling nEVs from each control group and the DNCB 14 days group, which showed the greatest changes in the expression of neuroinflammation markers in the hippocampus. Then, exosomal smRNA isolation and library preparation were conducted by Macrogen (Seoul, Korea), using the SMARTer smRNA-Seq Kit (Clontech Laboratories, Inc., Mountain View, CA, USA) according to the manufacturer’s instructions. Then, miRNA sequencing was performed by Macrogen, using the HiSeq 2500 System following the HiSeq 2500 System (User Guide Document #15035786 v02 HCS 2.2.70). Differentially expressed miRNAs were identified with a threshold p < 0.05.
Gene ontology (GO) analysis was performed to analyze the functional enrichment of differentially expressed miRNAs, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed to identify significantly enriched signaling pathways. Target genes for miRNAs showing significant changes in expression by AD were predicted using mirWalk, and all analyses of the predicted genes were performed using the database for annotation, visualization, and integrated discovery (DAVID) v6.8. *** p < 0.001 was applied as the criterion.

We performed NGS using neuronal exosomes isolated from the pooled serum of each Day 7 group (n = 6) and control group (n = 6), which showed the greatest difference in expression of neuroinflammatory markers in the hippocampus. Exosomal smRNA isolation and library preparation were performed by Macrogen (Seoul, Korea) using the SMARTer smRNA-Seq Kit (Clontech Laboratories, Inc., Mountain View, CA, USA) according to the manufacturer’s instructions. Subsequently, miRNA sequencing was conducted by Macrogen using the HiSeq 2500 system following the HiSeq 2500 system User Guide Document #15035786 v02 HCS 2.2.70. Differentially expressed miRNAs were identified with a threshold p < 0.05.
GO analysis was performed to analyze the functional enrichment of differentially expressed miRNAs, and KEGG pathway analysis was performed to identify significantly enriched signaling pathways. We used mirWalk to predict miRNA target genes that showed significant expression changes by stress, and then performed analysis. All the enrichment analysis was conducted using the database for annotation, visualization, and integrated discovery (DAVID) v6.8 (http://david.abcc.ncifcrf.gov/) (Access date: 20 May 2021). *** p < 0.001 was applied as the criterion.

The sequencing library was prepared using TruSeq Nano DNA Kit and sequenced with HiSeq 2500 System (2 × 100 paired-end) at Macrogen Inc. (Seoul, Republic of Korea). The resulting reads were filtered and trimmed to remove short and low-quality regions/reads and then assembled using CLC Genomics Workbench (CLC Bio, Aarhus, Denmark) with default parameters. The read mapping and genome annotation was performed using Geneious 7.1.8 [86 (link)]. ORFs were annotated using BLASTx search against the NCBI non-redundant protein database. Maximum likelihood (ML) tree reconstruction was performed with FastTree using the default options [87 (link)] on alignments of terminase genes and complete genomes (without gaps) from pbunaviruses (21 isolates/species). The branch support values were estimated with the Shimodaira-Hasegawa test [88 (link)]. The pairwise genome comparison was performed using PASC [50 (link)]. The genome of selected species and strains were compared using MAUVE [89 (link)] and plotted using genoPlotR package [90 (link)] available for R.