title: Small RNA Sequencing across Diverse Biofluids Identifies Optimal Methods for exRNA Isolation: Sample LUHomebrewF2
design: Small RNaseq libraries were prepared from 1.2 uL input RNA using the NEBNext Small RNA Sequencing Library Preparation kit (New England BioLabs), using a mosquito HTS automated nanoliter liquid handler (TTP Labtech). For the automation, the reaction volume was reduced to 1/5th of the manufacturer's recommended volume and the adaptors were diluted to 1/6th of the manufacturer's recommended concentration. Libraries were then cleaned and concentrated using the Zymo DNA Clean and Concentrator-5 kit (Zymo Research) with a 25 uL elution volume and quantified using the Quant-iT Picogreen DNA Assay High Sensitivity kit (ThermoFisher). The size distributions of the library products were determined using the Agilent High Sensitivity DNA chip on the Agilent 2100 Bioanalyzer instrument. The libraries were then pooled (up to 48 samples/pool) based on their concentrations and their size distribution, to obtain similar numbers of miRNA reads among libraries. The pooled libraries were then subjected to size selection on the PippinPrep instrument to remove unincorporated adapters and primers and adaptor-dimers.