Fast advanced master mix

Total RNA was extracted from cells in the lacrimal glands of the mice using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Complementary DNA was produced from total RNA using Superscript VILO™ Master Mix (Invitrogen). Quantitative real-time PCR was performed using the StepOne-Plus Real Time PCR system (Applied Biosystems) with Fast Advanced Master Mix (Applied Biosystems) and the predesigned primers for tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [TaqMan Gene Expression Assay (TNF-α: Mm00443258-m1, IL-6: Mm00446190-m1, and GAPDH: Mm99999915-g1)]. The mRNA levels were evaluated by the ΔΔCT method, and normalized to GAPDH mRNA.

Total RNA was isolated from THP‐1 cells (n = 16), “young M0” (n = 32), “aged M0” (n = 40), and M2 macrophages (n = 60) using the RNeasy purification kit (Qiagen, Germantown, MD, USA) and quantified with spectrophotometry (NanoDrop 1000, Thermo Scientific, Waltham, MA, USA). The sample sizes represent individual wells of cells harvested and are the result of the experimental setup (Figure S1 and S2). The high‐capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA) was used with 20 ng of total RNA to perform reverse transcription to cDNA according to the manufacturer's protocol. TaqMan PCR was run with TaqMan gene expression assays (CCL18: HS00268113_m1, CCL22: HS01574247_m1, CD80: HS01045161_m1, CD206/MRC‐1: HS00267207_m1, CXCL10: HS00171042_m1, IL‐1β: HS01555410_m1, IL‐6: HS00174131_m1, CXCL8/IL‐8: HS00174103_m1, IL‐10: HS00961622_m1, TNFα: HS00174128_m1, RNA18S5: Hs03928990_g1, MMP2: HS01548727_m1, MMP7: HS01042796_m1, MMP9: HS00957562_m1, MMP12:HS00159178_m1, ABHD5: HS01104373_m1, ADAMTS1: HS00199608_m1, AP‐1: HS00171851_m1, MGLL: HS00996004_m1, TLR4: HS00152939_m1) (Applied Biosystems) and Fast Advanced Master Mix (Applied Biosystems) using StepOne Real‐Time PCR systems (Applied Biosystems). Results for each target gene were normalized to 18S as the housekeeping gene and are given as mean ΔCT values.

Total RNA was extracted from the lacrimal gland or conjunctiva of mice by using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Complementary DNA was produced from total RNA using RevertraAce Master Mix (Toyobo, Osaka, Japan). Quantitative real-time (qRT-)PCR was performed using the StepOne-Plus Real Time PCR system (Applied Biosystems, Foster City, CA, USA) with Fast Advanced Master Mix (Applied Biosystems) and using the predesigned primers for mucin5AC (MUC5AC), fatty acid synthase (FASN), low density lipoprotein receptor (LDLR), uncoupling protein-1 (UCP-1) and beta-actin [TaqMan Gene Expression Assay (MUC5AC: Mm01276718-m1,FASN: Mm00662319_m1, LDLR: Mm01177349_m1,UCP-1: Mm01244861_m1 and beta-actin: Mm00607939-s1)]. The mRNA levels were evaluated by the ΔΔCT method, and normalized to beta-actin mRNA.

We used results of WGS to design a modified PCR assay targeting the tp0858 gene. We designed 2 new reverse PCR primers with different lengths and annealing temperatures, denoted “modified PCR-1” (5ʹ-GTGCGGTGAGCCCGGCGTT-3ʹ) and “modified PCR-2” (5ʹ-GTGAGCCCGGCGTT-3ʹ). We tested the assay using both gDNA from a Solomon Islands sample (WP0022.7liq) and from a non–Solomon Islands sample (non-SI, strain Gauthier). Double-distilled water was used as a control. We selected the shorter modified PCR-2 reverse primer for a modified qPCR assay. We performed qPCR using the original forward and reverse (denoted “2015 CDC RT-PCR” in Figure 2) 2015 CDC real-time PCR primers [8 (link)], as well as the original forward primer plus the modified PCR-2 reverse primer (denoted “modified PCR-2” in Figure 2). We also used the forward primer plus both reverse primer variants together (Figure 3; for conditions, see Supplementary Appendix). The PCR was run on an Applied Biosystems StepOne Plus qPCR machine using the TaqMan Fast Advanced Master Mix. We used a probe that detects both Solomon Islands and non–Solomon Islands strains (5ʹ-FAM-GCTGCAAGGAGAAGTCCTGCTGC-TAMRA-3ʹ).

Gene expression was assessed in 2, 7, and 21 day-cultured OA and healthy MSC-EV-treated pellets and control chondrocyte pellets. Total RNA was isolated and reverse transcribed with the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). qRT-PCR was performed using the Taqman assays (ThermoFisher Scientific; Table 1) and Fast Advanced Master Mix (Applied Biosystems, ThermoFisher Scientific) in a QuantStudio 3 Real-Time PCR system (Applied Biosystems). Genes for the analysis were selected based on the literature evidence of their involvement in the chondrogenesis and chondrocyte catabolic and anabolic activity [12 (link), 48 (link)] and are shown in Table 1.