16S sequencing of mouse stool from diversity outbred (DO) cohort 2 after exercise. 16S sequencing of stool from germ free (GF) mice with FMT from SPF mice, SPF mice treated with Neomycin, SPF mice treated with ampicillin.
Stool collected into 2ml tubes was snap frozen and stored in -80C until DNA extraction, that was perfomed by using DNeasy PowerSoil Kit (Qiagen, Cat. No. 12888-100) according to manufacturer instruction. DNA was amplified by using the KAPA Hifi HotStart ReadyMix (Roche, KK2602) and 16S specific primers spanning the variable region 1 and 2 (V1/V2) of the 16S rRNA gene were generated by using the following barcoded primers: Fwd i27- TATGGTAATTGTAGAGTTTGATCCTGGCTCAG, Rev 338-XXXXXXXXXXXXTGCTGCCTCCCGTAGGAGT, where each X represents a barcode base. Upon PCR amplification was performed the library was subsequently sequenced using 500 bp paired-end sequencing (Illumina MiSeq).