Fisherbrand commode specimen collection system

Participants were provided with a Fisherbrand™ Commode Specimen Collection System (FisherScientific, Waltham, MA, USA) to take home and asked to return their samples, preserved in RNAlater™ Stabilization Solution (ThermoFisher Scientific, Inc., Vilnius, Lithuania), within a week of blood collection. Samples were stored at −20 °C until ready for extraction. To extract stool DNA and RNA, we used a MagMAX Microbiome Ultra Nuclei Kit on a KingFisher Duo Prime Purification System following the manufacturer’s protocol (ThermoFisher). Briefly, we combined 250 μL from stool aliquots with 800 μL of Lysis Buffer, and vortexed the tube upside down for 10 s. Samples were placed on a shaker for 10 min at maximum speed (~2500 rpm). Then, 96-well plates were prepared according to the manufacturer’s protocol. Samples were centrifuged for 2 min at 14,000× g and 400 μL of the supernatant was transferred into row H of the prepared plate. We then added 520 μL of Binding Bead Mix ((500 μL Binding Buffer + 20 μL magnetic beads)/sample) to the samples and placed the plate in the KingFisher System. We ran the MagMAX Microbiome Ultra Nuclei program for RNA/DNA extraction and transferred the eluted samples into Eppendorf tubes stored at −80 °C.

Fecal samples were collected using the Fisherbrand™ Commode Specimen Collection System (Thermo Fisher Scientific, Waltham, MA, United States). Saliva samples were collected by asking each subject to let saliva collect in their mouth for at least 1 min. The subject was then asked to drool into a labeled 50 ml collection tube (Falcon, sterile conical polypropylene tube with flat-top screw cap). This process was repeated multiple times to collect larger volumes of saliva (2–5 ml). Oral cavity microbiome samples were from the dorsum of the tongue using Catch-All™ Sample Collection Swabs and swabbing 1 cm² of the center of the tongue for 5 s. Immediately after swabbing, each swab was swirled in MO BIO’s PowerSoil DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, CA, United States) collection tube with 750 ul prefilled collection buffer (Tube C1). The swab sponge was pressed against the tube wall multiple times for 20 s to ensure the transfer of bacteria from the swab to the solution. The specimen in the collection tube was kept cold until ready for processing.