Minocycline model
Bacterial 16S rRNA V4 fragments were PCR amplified using NEBNext Q5 Hot Start HiFi PCR master mix (NEB, U.K.) with universal 16S rRNA V4 primers [564F (TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-AYTGGGYDTAAAGNG) and 806R (GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-TACNVGGGTATCTAATCC)] with Illumina adaptor sequence overhangs included using the PCR cycle [denaturation (95 C x3 min for 1 cycle), amplification (95 C x30 sec, 50 C x30 sec, 72 C x30 sec, for 28 cycles) and final elongation (72 C x5 min for 1 cycle)]. PCR products were cleaned using AxyPrep Magentic beads (Axygen, U.K.) and the 16S fragments checked using gel electrophoresis and Agilent 2200 TapeStation nucleic acids system (Agilent Genomics, U.K.) before running a PCR for addition of the index sequences [denaturation (95 C x3 min for 1 cycle), amplification (95 C x30 sec, 55 C x30 sec, 72 C x30 sec, for 8 cycles) and final elongation (72 C x5 min for 1 cycle)]. The fragments were cleaned and quantified (as before) and samples sequenced using MiSeq sequencer (Illumina) with 250 bp paired-end reads.