16S seq of rat gut microbiome
Bacterial profiles were determined by broad-range amplification and sequence analysis of 16S rRNA genes in two sequencing runs following previously our described methods (Hara et al., 2012; Markle et al., 2013). In brief, amplicons were generated using primers that target the V3V4 variable region of the 16S rRNA gene: primers 357F (5-CCTACGGGAGGCAGCAG) and 806R (5-GGACTACHVGGGTWTCTAAT). PCR products were normalized using a SequalPrepTM kit (Invitrogen, Carlsbad, CA), pooled, lyophilized, purified and concentrated using a DNA Clean and Concentrator Kit (Zymo, Irvine, CA). Pooled amplicons were quantified using Qubit Fluorometer 2.0 (Invitrogen, Carlsbad, CA). The pool was diluted to 4nM and denatured with 0.2 N NaOH at room temperature. The denatured DNA was diluted to 15pM and spiked with 25% of the Illumina PhiX control DNA prior to loading the sequencer. Illumina paired-end sequencing was performed on the Miseq platform with v2.4 of the Miseq Control Software and of MiSeq Reporter, using a 600 cycle version 3 reagent kit. This resulted in sequences in a 2 x 300 paired-end format.