RNA-seq of human: saliva
Small RNA library preps were prepared using the QIAseq miRNA Library Kit (QIAGEN). All protocol steps were followed based on the use of the miRNeasy Serum/Plasma kit used upstream for purification of RNA. The recommended starting amount of total RNA is 5 ul of the RNA eluate when 200 ul of sample has been processed using the miRNAeasy Serum/Plasma Kit. Adapter dilutions throughout the protocol followed the Serum/Plasma recommendations. Cycles of library amplification followed that of a 10ng input sample with 19 total cycles. Two RNA control samples (Human XpressRef RNA, QIAGEN) with an input of 10 ng RNA were processed alongside the saliva samples. Final libraries were validated by Qubit Fluorometer (Invitrogen) and Fragment Analyzer (Agilent Technologies, Inc), and quantified via qPCR using NEBNext Library Quant Kit for Illumina (New England BioLabs, Inc). Pooled libraries were diluted, denatured and loaded onto the Illumina NextSeq 550 System, following the NextSeq User Guide. All 42 libraries were sequenced on one NextSeq High Output flow cell, single read 75 cycle run. FastQ files were developed for downstream analysis.