title: AMR-microbial-community
design: RNA was converted to cDNA using ProtoScript II First Strand cDNA Synthesis Kit (New England Biolabs). The V4 region of the 16S rRNA gene was amplified in triplicate for each sample using the 515F (44) and 806RB (45) Earth Microbiome primers and thermocycler protocol (46) in 25-l reactions containing Phusion High-fidelity Master Mix (New England Biolabs, Ipswich, MA), 0.25M of each primer, 3% dimethyl sulfoxide (as recommended by the manufacturer of the polymerase), and 2 l of DNA template. Triplicate reactions were consolidated and cleaned with a MinElute PCR purification kit (Qiagen) and quantified with a DS-11 FX+ spectrophotometer (DeNovix, Wilmington, DE). The pool of amplicon libraries was submitted to the University of Florida Interdisciplinary Center for Biotechnology Research (RRID:SCR_019152) for sequencing on an Illumina MiSeq with the 2x150bp v.2 cycle format.