Cs 2 sponges

Saliva samples were collected from infants using assisted-collection sponges and kits (CS-2 sponges and OG-250 kits) and from mothers using passive drool collection tubes (OG-500 kit) from DNA Genotek, Ottawa, Canada. Samples were stored at room temperature until DNA isolation. DNA was isolated using the manual purification protocol from DNA Genotek. Genotyping of CD38 rs3796863 was performed with a 5ʹ-nuclease assay using primers and probes from Applied Biosystems (TaqMan® SNP Genotyping Assay). Polymerase Chain Reaction was conducted with HotStarTaq Plus DNA Polymerase and Q-solution (Qiagen, Venlo, Netherlands) in a BioRad C1000 thermocycler with a CFX96 fluorescence reading module, using the following thermal protocol: denaturation at 95°C for 5 min; followed by cycling: 95°C for 15 s and 60°C for 1 min for 45 cycles.

Twenty-eight additional Malawian DC buccal samples had both DNA and RNA isolation, and these were used for gene expression-methylation correlation analysis. DNA was extracted as outlined above, and methylation was assessed using the Illumina EPIC methylation kit in 24 samples following procedures as outlined for the main dataset. The Oragene RNA (RE-100) RNA collection kit was used in conjunction with CS-2 sponges (DNA Genotek Inc., Ottawa, Ontario, Canada) with modification, to collect buccal epithelial cells from multiple cheek swabs. RNA was extracted according to the manufacturer’s instructions. Gene expression analysis was undertaken for 24 samples using the Affymetrix GeneChip human Clariom S assay (Thermo Fisher Scientific, Waltham, Massachusetts, USA) following the recommendations of the GeneChip WT PLUS Reagent Kit—Target Preparation for GeneChip Whole Transcript (WT) Expression Arrays manual.

Passive drool was collected from infants at 5 months of age using CS-2 sponges and OG-250 kits (DNA Genotek, Ottawa, Canada) and was stored at room temperature until DNA isolation. We incubated collection kits at 50 °C for 1 h, then centrifuged for 10 min at 200 rcf to release all liquid from sponges. We isolated DNA from 500 μL of saliva using the manual purification protocol from DNA Genotek. DNA was stored in Hydration Solution (10 mM Tris, 1 mM EDTA, pH 7–8, Qiagen, Valencia, CA) and quantitated using nanodrop. One infant was excluded from the final analysis due to insufficient DNA.