title: RNA-Seq of mouse gut metagenome: high dose GRP
design: Genomic DNA was extracted from the fecal samples by using the EZNA Soil DNA kit. DNA quality was verified by 1% agarose gel electrophoresis and a Nanodrop 2000. The bacterial 16S rRNA V3V4 region was amplified via PCR by using FastPfu polymerase and the primers 338F (5'-TCCTACGGGAGGCAGCAGT-3') and 806R (5'-GGACTACCAGGGTATCTAATCCTGTT-3') under the following conditions: 95 for 3 min; 27 cycles of 95 for 30 s, 55 for 30 s, and 72 for 45 s; and 72 for 10 min. PCR was performed in triplicate, and the products were pooled, purified (AxyPrep DNA Gel Extraction Kit), and quantified by using a Quantus Fluorometer. Purified amplicons were sequenced on an Illumina NextSeq 2000 platform. The sequences were quality-filtered, clustered into operational taxonomic units (OTUs) at 97% similarity (82% confidence) and analyzed across taxonomic levels.