16S sequencing of cohoused young and old mice

The goal of this experiment was to decouple host and microbiome age. To do this, we obtained n=25 8-week-old female C57BL/6 mice from The Jackson Laboratory and n=20 19- and 20-month- old female C57BL/6 mice from the National Institutes on Aging. Prior to the start of the experiment, mice were housed five per cage with other mice of the same age. Mice were allowed to acclimate for at least one week prior to the start of the experiment. During cohousing, three young mice were housed with two old mice for one month. Control young and old mice remained housed with other mice of the same age. After one month, two of the cohousing cages were separated by age and two cohousing cages remained cohoused. Stool pellets were collected at baseline, after one month of cohousing, and after two, four, six, and eight weeks of separation. DNA was extracted from stool pellets 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. Note that, due to a machine failure, the second MiSeq run failed at cycle 428, so R2 for the second MiSeq run is 164 nucleotides instead of 250.