title: metagenomics of Homo sapiens: adult diarrheal stool
design: Total DNA and RNA were extracted from samples using the ZymoBIOMICS DNA/RNA Miniprep Kit (Zymo Research, Irvine, CA, USA) following the manufacturer protocol with modifications for RNA: (i) one volume of lysis buffer was used, (ii) the optional DNase I treatment was performed, and (iii) RNA was eluted with 50  of DNase/RNase-free water. After extraction, DNA and RNA quantities were measured using a Qubit Flex Fluorometer with the DNA and RNA High Sensitivity Assay Kits (Thermo Fisher Scientific, Waltham, MA, USA). Fecal DNA was prepared for metagenomic sequencing using the Illumina Nextera XT DNA Library Preparation Kit, following the manufacturer protocol. Libraries were indexed, amplified, and purified according to the standard tagmentation workflow. Total RNA was used to generate metatranscriptomic libraries with the NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA), which employs a random priming approach for reverse transcription. The protocol for purified mRNA was followed.  Library fragment size distributions were assessed using an Agilent 2100 Bioanalyzer with the High Sensitivity DNA kit. Results from these steps were used to pool samples in an equimolar ratio. Libraries were pooled in equimolar amounts, purified with SPRI beads at a 0.7x ratio, and sequenced on the Element Biosciences Aviti platform (2 x 150 bp) at the UC Davis Genome Sequencing Center (Davis, CA).