16S sequencing of cohoused germ-free mice that received FMTs from young or old donors

We performed cohousing of young germ-free (GF) mice after they had received fecal microbiome transplants (FMTs) from young or old donor mice. For donor material, we collected stool pellets from young (3 months) and old (17 months) female C57BL/6 mice. The recipients were young (3-5 months) GF female C57BL/6 mice. After receiving the FMT, recipients were housed for 2 weeks in isocages with other mice that received the same FMT. Then, we cohoused recipients of young microbiomes (3 per cage) with recipients of old microbiomes (2 per cage). Non-cohoused control cages contained 5 young FMT recipients or 4-5 old FMT recipients. After two weeks and one month of cohousing, we collected stool pellets from all recipients, and performed 16S sequencing. DNA was extracted using the Qiagen DNeasy PowerSoil Pro Kit, the V1/V2 variable region of the 16S gene was amplified using KAPA HiFi HotStart ReadyMix and the F27/R338 primer-pair (F27: 5'-AGAGTTTGATCCTGGCTCAG-3', R338: 5'-TGCTGCCTCCCGTAGGAGT-3'), pooled libraries underwent size selection using AMPure XP SPRI beads, and pooled libraries were paired-end (2x250 bp) sequenced across two MiSeq runs.