Smarter smrna seq kit

RNA was isolated using Trizol or LS Trizol (Invitrogen). Total RNA samples were fractionated into small RNA (<200 nt) and long RNA (>200 nt) using the Monarch RNA cleaner (New England Biolabs). Small RNA libraries were prepared using the Small RNA-Seq Library Prep Kit (Lexogen). Both 5’-phosphate and 5’-phosphate independent libraries (RppH-treated)^{124 (link)} were prepared. Libraries from 4-cell embryos were also prepared using 18–30 nt gel-purified RNA using the SMARTer smRNA-Seq Kit (Clontech) and NEXTflex-Small-RNA-Seq (New England Biolabs) with similar results. RNA > 200 nt was treated with TURBO DNase (Ambion) and rRNA depletion carried out using RiboCop rRNA Depletion Kit for Human/Mouse/Rat (Lexogen). Long RNA (>200 nt) libraries were made using CORALL Total RNA-Seq Library Prep Kit (Lexogen). Both small and long RNA libraries were sequenced 150 nt from both ends using the Illumina NovaSeq 6000 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.

The total RNA was isolated from kidney tissue, serum EVs and urinary EVs using the miRNeasy Mini kit (Qiagen, Venlo, The Netherlands) following the manufacturer’s instruction. Tissue samples were treated with QIAzol and mechanically homogenized before the RNA extraction.
The RNA quality and concentration were checked using the Agilent 2100 bioanalyzer. The miRNA sequencing was performed using the NGS sequencing Illumina NextSeq 500 platform with the SMARTer^® smRNA-Seq kit (Clontech, Mountain View, CA, USA). As a positive control, we used a known microRNA: miR-163s.

Libraries of small RNA were prepared using three different protocols: (i) NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England BioLabs, Ipswich, MA, USA); (ii) NEXTFlex Small RNA-Seq Kit v3 (PerkinElmer, Waltham, MA, USA); and (iii) SMARTer smRNA-seq kit (Clontech Laboratories, Mountain View, CA, USA), according to the manufacturer’s guidelines. The amount of RNA was 0.7 ng for the plasma 1 sample and 1 ng for the plasma 2 sample. The size profiles of the individual libraries were analyzed using a high sensitivity DNA assay on a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). Size selection of fragments was carried out using 5% Mini-PROTEAN TBE Gel pre-cast polyacrylamide gel (Bio-Rad Laboratories, Hercules, CA, USA). Electrophoresis was performed at 120 V for 1 h in tris-borate-EDTA (TBE) buffer. The band of interest was eluted, following the NEXTFlex kit guidelines. Libraries were quantified by qPCR using KAPA Sybr fast master mix (2X) optimized for LightCycler 480 (KAPA Biosystems, Wilmington, MA, USA) and an in-house standard library from Fundación Parque Científico de Madrid. Quantified libraries were mixed at equimolar ratios and sequenced with a NextSeq 500/550 High Output Kit v2.5 (75 Cycles) using the NextSeq 500 system (Illumina, San Diego, CA, USA).