16S metagenomics of Mus musculus: faecal sample
Faecal DNA was isolated using QIAamp PowerFecal DNA KIT (Qiagen, #12830-50). 16S amplicons were prepared for sequencing by indexing PCR by a reduced-volume reaction based on the Illumina 16S Metagenomics Sequencing Protocol (15044223 Rev. B). Briefly, 2 L of each sample was combined with 3.25ul H2O, 6.25ul of 2xNEB Q5 High-Fidelity DNA Polymerase 2X Master Mix (M0492L), and 1ul of a unique 10M UDI primer from the set Nextera IDT-8nt (384 Indexes). Samples were incubated at 95C for 3 m, followed by 10 cycles of 95C for 30 s, 55C for 30 s,72C for 30 s, followed by a final extension at 72C for 5 m. A bead-based cleanup was carried out with Beckman Coulter SPRIselect (B23319) in a 0.8x ratio. The quality of the purified libraries was assessed using a D1000 ScreenTape on an Agilent 24200 TapeStation. Libraries were sequenced on the Illumina MiSeq platform. 2x300 paired-end reads were produced using the 600 cycle MiSeq Reagent kit v3. Libraries were loaded at 8pM concentration with 20% PhiX.