Nanodrop

The HCT116 cells were seeded in a T25 flask and treated with GIV. RNA was isolated from the cells using total RNA isolation kit (Promega, REF Z6111). RNA was quantified using Nanodrop, and 1 µg of the total RNA was used to synthesize cDNA using the cDNA synthesis kit (Promega, REF A3800). The cDNA obtained was used as a template to check the expression of apoptosis-associated genes, such as Caspase3, Caspase9, Bcl2, and Parp (primers were purchased from Imperialls, ILS) using SYBR Green Master Mix (Promega, REF A6001). 18s rRNA was used as an internal control. The Ct values attained through q-PCR were used to analyze the expression of the genes (Jozefczuk and Adjaye, 2011 (link)).

RNA was extracted from cell extracts using an RNA easy mini-kit (Qiagen, UK) as per manufacturer’s instructions. RNA concentration was then quantified using a Nanodrop and 1 μg of RNA was used for cDNA synthesis using the Promega ImpromII kit. The SYBR real-time PCR system (Kapa Biosystems, UK) was used to quantify transcript abundance for genes of interest and18S mRNA was used as a control. Template equivalent to 5 ng of RNA in cDNA library per reaction was added to each 20 μl reaction with a final primer concentration of 200 nM per reaction. Crossing thresholds were determined using MxPro software (Agilent), and fold-difference in RNA quantity was calculated using the relative quantification method (2^-ΔΔct).

RNA was extracted from leaves, stems and roots of 2-month old Hamlin and Carrizo using Trizol reagent according to the manufacturer's instructions (Sigma-Aldrich, St. Louis, MO). Total RNA was quantified using the Nanodrop and treated with RQ1 RNase-free DNase from Promega Corp (Madison, WI). DNase-treated RNA (~1.5 μg) was used to synthesize first-strand cDNA with 0.5 μg of oligo (dT) primer and 1 μL of SuperScript® III reverse transcriptase in a 20 μL reaction (Invitrogen). A negative control without the reverse transcriptase was performed to verify absence of genomic DNA contamination. RT-qPCR was run and analyzed on a real time PCR machine ABI7500 using cDNA as described (Hao et al., 2016 (link)). Primers for CsthiF, Csthi1, and Csthi2 are listed in Table 1. The citrus gene Glyceraldehyde-3-phosphate dehydrogenase C2 (GAPC2) was amplified with primers CsGAPC2-5′ and CsGAPC2-3′ and used to normalize the values as an internal control (Table 1). The gene expression levels in leaves, stems and roots of Hamlin and Carrizo were compared by 2^ΔCt fold which was obtained for each sample versus a reference sample which had the lowest 2^ΔCt in the tested samples. ΔCt = Ct (internal control) - Ct (target gene). The qPCR reactions were set up in triplicate and repeated twice with similar results.

Genomic DNA was extracted from peripheral blood samples using standard methods and stored at −80°C until further use. Prior to sequencing gDNA concentration was determined using nanodrop (at OHSU) and QuantiFluor dsDNA System by Promega (at NEI), according to the manufacturer’s instructions. WES DNA libraries (from DNA samples of individuals selected for WES) and sequencing was done in the Cancer Genomics Research Laboratory (CGR), Leidos Biomedical Research, Frederick National Laboratory for Cancer Research, Frederick, MD, USA using standard protocols. Pair-ended 149bp sequencing was performed on NovaSeq 6000 (Illumina, Inc., San Diego, CA).

RNA from kidney tissue was extracted with TriReagent. Briefly, RNA was precipitated from the aqueous phase with isopropanol and washed with 70% ethanol. Purity was assessed using a Nanodrop and cDNA synthesis was performed (Promega RT kit). Real time PCR was performed with SYBR Green JumpStart ready mix according to manufacturer’s instructions. Data was calculated using ∆∆Ct and 18S was used as a housekeeping gene. Data is plotted against vehicle control group. Primer sequences used are reported in the Supplementary Table S3.