title: Metatranscriptome of T3
design: RNA were sequentially extracted from sample swabs using the ZymoBIOMICS DNA/RNA Miniprep kit (Zymo Research) along with positive and negative controls. The positive controls were standardized mock communities from two different manufacturers (ATCC and Zymo Research). The ATCC mock community was an equal mixture of 10 yeast and fungi (Mycobiome Whole Cell Mix, ATCC MSA-2010) and the Zymo Research mock community was an equal mixture of bacteria and yeasts (ZymoBIOMICS Microbial Community Standard D6300).  The negative control was a blank swab.  After extraction RNA concentrations were assessed using the Qubit RNA assay. RNA integrity (RIN) was determined using the High Sensitivity RNA ScreenTape (5067-5579) assay on a TapeStation (Agilent). Extracted RNA was converted to double stranded cDNA using the Maxima H Minus Double-Stranded cDNA Synthesis Kit (ThermoFisher). Libraries of double stranded cDNA were then prepared using the Nextera DNA Flex kit (Illumina) with Unique Dual Indices (Illumina). Library concentration was then assessed using the QuBit dsDNA assay and insert size determined using the High Sensitivity RNA ScreenTape kit on a TapeStation (Agilent). Finally, 25 l of a 10nM library were then sent to the University of Kansas Medical Center  and sequenced on a NovaSeq6000, using an S1 flowcell with 300 cycle chemistry.