16S sequencing of stool from single antibiotics treated mice (neomycin, ampicillin, vancomycin, metronidazole, imipenem, and ciprofloxacin)
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).