Dsdna qubit

All samples were analyzed in a Clinical Laboratory Improvement Amendments certified laboratory using clinically validated methods. Samples were processed by liquid handlers in a 96-well format. Each batch of 96 samples included external positive and negative process controls, to ensure acceptable method performance. The samples underwent mechanical and chemical lysis and library preparation as previously described [Citation37]. Briefly, the RNA was extracted using silica beads and a series of washes, followed by elution in molecular biology grade water. DNA was degraded using RNase-free DNase. Prokaryotic and human rRNAs were removed using a subtractive hybridization method: biotinylated DNA probes with sequences complementary to microbial and human rRNAs were added to total RNA, the mixture was heated and cooled, and the probe-rRNA complexes were removed using magnetic streptavidin beads. The remaining RNAs that contained all other human and microbial RNAs (coding and noncoding) were converted to directional sequencing libraries using unique dual-barcoded adapters and ultrapure reagents. Libraries were pooled and assessed for quality with dsDNA Qubit™ (Thermo Fisher Scientific, MA, USA) and Fragment Analyzer (Advanced Analytical, CA, USA) methods. Library pools were sequenced on an Illumina NovaSeq™ 6000 instrument (Illumina, Inc., CA, USA) using 300-cycle kits.