title: Shoreline StrainID, Sample Dog_9; Month: 0; Storage Temp: NA; Cell4; Plate BC1
design: We prospectively collected stool samples from 20 apparently healthy dogs following receipt of each owners written consent to participate in the study. The dogs had no history of gastrointestinal dysfunction and administration of antibiotics, immunomodulatory, or gastroprotectant drugs 6 months before enrollment. Fresh morning stool was collected within 2 hours of defecation. Stool samples were manually homogenized in cold sterile 1PBS, filtered through a metal sieve, and centrifuged at 600g for 10 minutes at 4, discarding the supernatant. Each fecal pellet was resuspended in 20% glycerol in PBS for cryopreservation. Aliquots of each resuspended stool sample were transferred to sterile cryogenic vials, snap-frozen in liquid nitrogen, and stored at -20 or -80 for 3, 6, 9, and 12 months. Additional aliquots from each dog were used as a baseline samples, and their DNA was extracted immediately (i.e., not frozen) following treatment with PMA. At 3-month, 6-month, 9-month, and 12-month, the frozen aliquots were thawed on a dry heating block at 22 for approximately 10 minutes and immediately treated with PMA before DNA extraction. To understand the effect of PMA treatment on fecal microbiome composition, feces samples from 10 of the dogs were pooled together in equal quantities and were aliquoted to 6 samples to serve as lysis/PMA controls. Fresh samples, and those where lysis was induced by exposure to heat (100 on a dry block for 5 minutes) and freeze-thaw (3 cycles) were subjected to DNA extraction following treatment with PMA or the DMSO (20%) diluent. Samples of glycerol, PBS, and nuclease-free water were submitted for sequencing to detect reagent contamination. 1 mg of PMA stock (Biotium Inc., Hayward, CA) was diluted in 1 mL 20% DMSO in sterile, DNA/RNA/nuclease-free water. 1 mL of each aliquot was diluted in 1PBS for a final volume of 5 mL and mixed with 110 L of diluted PMA (43M). To prevent light contamination, the samples were incubated with gentle tilting (6 rpm) at room temperature for 30 min in a styrofoam cooler lined with aluminum foil. The samples were then incubated for 20 minutes under full light from a LED aquarium light source affixed to the open cooler top. The samples were centrifuged at 600 g for 10 minutes at 4, and the supernatant was discarded. The pellets were washed with 1 mL 1 PBS, centrifuged at 600 g for 10 minutes at 4, and the supernatant was discarded. Bulk genomic DNA was purified from 200 mg of the fecal pellet using the QIAGEN Power Fecal Pro DNA kit (Qiagen Inc., Hilden, Germany) per the manufacturers protocol with an additional incubation step at 65 for 5 minutes before tubes were vortexed for 10 minutes.