title: 16S rRNA Seq of mus musculus : LB stool
design: The V4 hypervariable region of the 16S rRNA gene is amplified using                 a universal forward sequencing primer and a uniquely barcoded reverse sequencing                 primer to allow for multiplexing 90. Amplification reactions are performed using                 12.5L of KAPA2G Robust HotStart ReadyMix (KAPA Biosystems), 1.5L of 10M forward and                 reverse primers, 8.5L of sterile water and 2L of DNA. The V4 region was amplified by                 cycling the reaction at 95C for 3min, 24x cycles of 95C for 15s, 50C for 15s and 72C                 for 15s, followed by a 5 min 72C extension. All amplification reactions were done in                 triplicate, checked on a 1% agarose TBE gel, and then pooled to reduce amplification                 bias. Pooled triplicates were quantified using Quant-it PicoGreen dsDNA Assay                 (Thermo Fisher Scientific) and combined by even concentrations. The final library                 was purified using Ampure XP beads (Agencourt), selecting for the bacterial V4                 amplified band. The purified library was quantified using Qubit dsDNA Assay (Thermo                 Fisher Scientific) and loaded on to the Illumina MiSeq for sequencing, according to                 manufacturer instructions (Illumina, San Diego, CA). Sequencing was performed using                 the V2 (150bp x 2) chemistry.