title: Murine.metagenome.dysbiosis.short
design: Amplicon library  preparation was performed using an automated platform (Biomek 4000) using a  custom liquid handling method. For each sample, the V4 region of the  bacterial 16S rRNA gene was amplified in duplicate reactions, using primer  set 515F/806R, which nearly universally amplifies bacterial and archaeal 16S  rRNA genes.1,2 Each unique barcoded amplicon was generated in pairs of 25_l  reactions with the following reaction conditions: 11_l PCR-grade H2O, 10_l  Hot MasterMix (5 Prime Cat# 2200410), 2_l of forward and reversed barcoded  primer (5_M) and 2_l template DNA. Reactions were run on a C1000 Touch  Thermal Cycler (Bio-Rad) with the following cycling conditions: initial  denaturing at 94C for 3 min followed by 35 cycles of denaturation at 94C for  45 seconds, annealing at 58C for 1 minute, and extension at 72 C for 90  seconds, with a final extension of 10 min at 72C. Amplicons were quantified  using Agilent 2200 TapeStation system and pooled. Purification was then  performed using Ampure XT (Beckman Coulter Cat# A63882) as per the  manufacturer instructions