title: A4 microbiome 20d
design: All samples were processed and sequenced by the University of Illinois s Roy J. Carver High-Throughput Sequencing and Genotyping Unit (Urbana, IL). In brief, DNA concentrations were measured via a Qubit instrument (ThermoFisher) and diluted to a final concentration of 2 ng/ l. Reaction mixes contained the Fast Start High Fidelity PCR System (Roche, Basel, Switzerland) and 20x Access Array loading reagents (Fluidigm, San Francisco, CA) per the manufacturer s protocol. For 16S rDNA amplification, the following bacterial primers were used: 515FB-FWD: GTGYCAGCMGCCGCGGTAA and 806RB-REV: GGACTACNVGGGTWTCTAAT. PCR was performed using standard conditions (Parada et al., 2016). PCR amplicons were size selected on a 2% agarose E-gel (ThermoFisher) and purified via a gel extraction kit (Qiagen, Hilden, Germany). Amplicon sizes were then verified post purification using a Bioanalyzer (Agilent, Santa Clara, CA).