Fragment bioanalyzer system with dna 1000 chip

8 µl of bdEV (from a total of 40 μl RNAs) and 20 ng of BH RNAs were used for small RNA library construction by the D-Plex Small RNA-seq Kit (Diagenode C05030001). D-Plex Single Indexes for Illumina - Set A (DiagenodeC05030010) were attached according to the manufacturer’s protocol. The yield and size distribution of the small RNA libraries were assessed using the Fragment Bioanalyzer^™ system with DNA 1000 chip (Agilent5067–1505). 170–230 bp libraries were selected with agarose gel cassettes (Sage Science HTG3010) on the BluePippin Size Selection System (Sage Science). Multiplexed libraries were equally pooled to 1nM and sequenced on the NovaSeq 6000 system (Illumina) with the NovaSeq 6000 SP Reagent Kit v1.5 (100 cycles) (Illumina 20028401).

1 µg of BH RNAs was incubated with 1 U/µg RNase R (Lucigen RNR07250) at 37°C for 30 min. RNase R-treated RNAs were then re-isolated by RNA Clean & Concentrator^™−5 (Zymo Research R1014) according to the manufacturer’s protocol. 100 ng of total BH RNA with and without RNase R treatment was then used to construct cDNA libraries by Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus (Illumina 20072063). A Ribo-Zero Plus kit was used to deplete ribosomal RNA in these samples, and library construction was then done per manufacturer’s protocol. Indexes were attached using IDT for Illumina RNA UD Indexes according to the manufacturer’s protocol. The yield and size distribution of the total RNA libraries were assessed using the Fragment Bioanalyzer^™ system with DNA 1000 chip (Agilent 5067–1505). Multiplexed libraries were equally pooled to 1nM and sequenced with the NovaSeq 6000 system (Illumina) and NovaSeq 6000 S2 Reagent Kit v1.5 (300 cycles) (Illumina 20028314).