Nanodrop one

For work at ZARI, Zambia: One representative plate with pure cultures was selected from each of the six triplicated samples (WS1-WS6) and lyophilized overnight at -40°C. The lyophilized cultures were scrapped off the plate into a mortar, to which 1 ml 2% CTAB extraction buffer containing 2% (wt/vol) CTAB, 2% PVP-40, 100 mM Tris-HCl (pH 8.0), 25 mM EDTA, 2M NaCl, and 2% mercaptoethanol (added immediately before use) was added. Using a pestle, the sample was macerated, followed by incubation at 65°C for 15 minutes. An equal volume of chloroform-isoamyl alcohol (24:1) was added to the sample, vortexed and then centrifuged at 12,000 rpm for 15 minutes. The rest of the extraction protocol was conducted as previously described [1 (link)]. Dried pellets were suspended in 25 μl DEPC treated water followed by quantification with NanoDROP ONE (Thermal Scientific, CA, USA).
For work at USDA-ARS, USA: A subset of samples WS4 and WS5 was shipped to USDA-ARS FDWSRU under permit from the United States Department of Agriculture–Plant Protection and Quarantine (P526P-19-02423) for further analysis. The two isolates were first single conidia purified, thus obtaining sub-isolates Zambia1.2 (WS4) and Zambia 2.1 (WS5). DNA was extracted from the two sub-isolates and isolates Rb3 (MoO), PL3.1 (MoL), T25 and B2 (MoT) as previously described [1 (link)].

Gene expression levels within the tibial tuberosity were calculated using the CFX96 Real-Time System (Bio-Rad, Hercules, CA, USA). Total RNA was extracted from the tibial articular cartilage using TRIzol (15596026; Thermo Fisher Scientific, Chiba, Japan). To ensure RNA purity (with a 260/280 ratio between 1.8 and 2.0) tRNA was analyzed using NanoDrop One (Termo Fisher). Subsequently, cDNA was synthesized using the iScript gDNA Clear synthesis kit (172530; Bio-Rad Laboratories, Hercules, CA, USA). The concentration of tRNA during cDNA synthesis was standardized to 10 ng/µl. Gene expression analysis was performed using the TaqMan probe assay. Furthermore, 45S ribosomal RNA was used as a housekeeping gene. The PCR conditions were as follows: initial denaturation at 95 °C for 20 s, then 40 cycles of denaturation at 95 °C for 15 s, and a final extension at 60 °C for 1 min. Rn45S mRNA was used as an internal control. The relative expression levels were calculated using the 2^−ΔΔCT method^{26 (link)}.

About 4 to 6 sterile paper points were inserted in the periodontal pockets for 30 s, removed and stored in 2mls cryo- vials containing 100 μl of DNA shield (Zymoresearch, CA, USA). The samples were then transported on ice to the Molecular Biology Laboratory of department of Anatomy, Makerere University College of Health Sciences. The laboratory is located within the histology laboratory on the ground and first floor of the department. DNA was extracted from these samples using the Quick-DNA Mini prep Plus Kit (Zymoresearch, CA, USA) as per the manufacturer’s instructions. It was then quantified by Nanodrop One (Themo Fisher Scientific, California, USA).

Total RNA was extracted from cell or tissue samples using an RNA-quick Purification Kit (Esunbio, Shanghai, China) as per the manufacturer’s instructions. Genomic DNA (gDNA) was extracted from cell samples using a Genomic DNA Extraction Kit (TIANGEN, Beijing, China). The density and quality of total RNA and gDNA were detected by NanoDrop One (Themo Fisher Scientific, Waltham, USA).

Total RNA was isolated from the calvariae using QIAzol (QIAGEN, Hong Kong, China), followed by quantification via NanoDrop One (Thermo Scientific). Further, cDNA synthesis was carried out using the high-capacity cDNA reverse transcription kit (Applied Biosystems, Waltham, MA, USA). RT-PCR analysis was performed for osteoclastogenic markers, like nuclear factor kappa B (NF-κB), matrix metallopeptidase 9 (Mmp-9), cathepsin K (Ctsk), nuclear factor of activated T-cell cytoplasmic-1 (Nfatc-1), and chemokine ligand 2 (Ccl-2), using SYBR Green PCR master mix (Bio-Rad, Hong Kong, China) with a CFX Connect RT-PCR detection system (Bio-Rad).

To understand the role of LBS6-induced expansion of cotyledons, real-time expressions of the genes involved in cell number, division rate, expansion, size, and phytohormonal regulation were carried out using specific primers designed from sequences retrieved from ENSEBL-Plants (Supplementary Table S2). For gene expression analysis, the etiolated, excised cotyledons were placed on filter paper moistened with 1ml/L of LBS6 and sterile double distilled water. The cotyledon samples were harvested after 0, 8, 24, 48 and 72 h. The samples were flash frozen in liquid nitrogen and stored at -80°C. Total RNA was extracted using Trizol (Takara Bio, USA), and quantified with NanoDrop One (ThermoScientific, USA). The cDNA was prepared using 1 µg of RNA with iScript cDNA synthesis kit (Bio-Rad, USA). Real-time qPCR analysis was performed in a Quant Studio5 (ThermoScientific, USA). β-tubulin was used as a reference gene. The specificity of PCR amplification was confirmed at the end of the PCR cycles by melt-curve analysis. Three independent experiments were performed with three replicates each, and the relative gene expression was calculated using the 2^-ΔΔCt method (Livak and Schmittgen, 2001 (link)).

RNA quantity was assayed using UV spectrophotometry on Nanodrop One (Thermo Scientific). Optical density absorption ratios A260/A280 & A260/A230 of the cerebellum samples were 1.86(±0.03 SD) and 2.29 (±0.07 SD) respectively. The corresponding values for the spinal cord samples were 1.95 (±0.03 SD) and 2.08 (±0.32 SD) respectively. Furthermore, RNA integrity was verified using denaturing formaldehyde agarose gel electrophoresis. All samples showed intact bands for 28S and 18S rRNA and were subsequently used for RTqPCR.