Nanodrop

100 mg fresh leaves transgenics were homogenized in liquid nitrogen and mixed with 500 μl CTAB buffer (2% w/v CTAB, 0.3 M NaCl, 20 mM EDTA, 100 mM Tris–Cl (pH-8) 0.2% BME). Genomic DNA was isolated according to Murray and Thompson³⁶ . Quality and quantity of genomic DNA was checked by gel electrophoresis and nanodrop (GE, US), respectively.
Transgene copy number was checked using 10 μg of genomic DNA. DNA was digested with BamHI and separated by 0.8% agarose gel electrophoresis. DNA was blotted on Hybond-N Nylon membrane (Pharmacia) and fixed using UV cross linker (UVP HybriLinker, Analytik Jena, US). Blot was probed with hpt gene probe which was synthesized using PCR DIG probe synthesis kit (SIGMA-ALDRICH). Hybridization and detection were done using the non-radioactive method by DIG Luminescent Detection Kit as per the manufacturer guidelines (SIGMA-ALDRICH) with hybridization temperature 42 °C and washing 65 °C. Detection was carried out using CSPD solution and visualized by Fujifilm LAS4000 luminescence imager.

Overnight cultures of the various S. mutans strains were adjusted to OD_600nm = 0.1 in BHI and incubated in the absence or presence of 1.25 µg/mL CBG and/or 21-CSP (1 µg/mL). After a 4 h incubation at 37 °C, the bacteria were resuspended in 1 mL RNA protect reagent (Qiagen, Hilden, Germany) for 5 min. Following centrifugation (1500× g, 15 min, 4 °C), the pellets were stored at −80 °C. On the day of RNA extraction, the pellets were resuspended in 350 µL RLT lysis buffer (Qiagen, Hilden, Germany), placed into microcentrifuge tubes containing 1 mm glass beads followed by beating 3 times at a speed of 4.5 m/s for 45 s at 5 min intervals using the Bio 101 FastPrep FP120 cell disruption system (Savant Instruments, Inc., Holbrook, NY, USA). The samples were further processed for RNA isolation using the RNeasy MINI kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions including an on-column DNase digestion step [35 (link)]. The RNA concentration and purity were analyzed by a Nanodrop (Nanovue, GE Healthcare Life Sciences, Buckinghamshire, UK). Samples that had an OD₂₆₀/OD₂₈₀ ratio of 1.8–2 and an OD₂₆₀/OD₂₃₀ ratio above 2 were used for cDNA synthesis. The RNA was reverse transcribed into cDNA using the qScript cDNA synthesis kit (QuantaBio, Beverly, MA, USA) [35 (link)].

The assay was performed similarly to the method used by Feldman et al. (2014) (link) with slight modifications. An overnight culture of V. harveyi (OD₅₉₅∼0.7) was diluted 1:10 in complete AB medium and incubated at 30°C under aerobic conditions in the absence or presence of 2 μg/ml CBG for 10 h. Eight milliliter of bacterial culture were used for each sample. At the end of incubation, the supernatant was removed, and the cells were washed with 2 ml of PBS and incubated with 2 ml of RNA Protect (Qiagen, Hilden, Germany) for 5 min at room temperature. RNA was isolated using the RNeasy MINI kit (Qiagen) including on-column DNase digestion according to the manufacturer’s instructions. RNA purity and quantity were determined using Nanodrop (Nanovue, GE Healthcare Life Sciences, Buckinghamshire, United Kingdom). Only samples with an OD₂₆₀/OD₂₈₀ ratio of 2 and an OD₂₆₀/OD₂₃₀ ratio above 2 were used for cDNA synthesis. The samples were stored at −80°C until use.

RNA extraction was performed using the Qiagen RNEasy kit according to the manufacturer’s instructions. Resulting RNA was incubated with DNase (Amplicon) for 15 mins before inactivation. RNA concentration was determined using a Nanodrop (GE Healthcare, UK). cDNA was synthesised using MMLV reverse transcriptase (Promega) according to manufacturer’s instructions. Primers were custom designed across exon boundaries using the Primer3 web-based program (http://primer3.ut.ee/) and purchased from Sigma. cFLIP: Forward: 5′-TGATGGCAGAGATTGGTGAG-3′, and reverse 5′-GATTTAGACCAACGGGGTCT-3′. Axin 2: Forward, 5′-AGTGTGAGGTCCACGGAAAC-3′ and reverse 5′-TGGCTGGTGCAAAGACATAG-3′. qPCR was performed using GoTaq polymerase (Promega) and RealTime PCR machine using StepOne software (Applied Biosystems).

Genomic DNA from the spleens was extracted by traditional phenol and chloroform methods. The quality of DNA was evaluated by 0.8% agarose gel electrophoresis using 100 ng extracted DNA. The concentration was detected by NanoDrop (GE Healthcare, USA) spectrophotometer, and then a total of 1.5 μg genomic DNA from each sample was treated with sodium bisulfite using an EZ DNA Methylation-Gold Kit (Zymo Research, Orange, CA) according to the manufacturer's protocol.

Total genomic DNA extracted from plants was quantified using a NanoDrop spectrophotometer, adjusted to a concentration of 50 ng/μl, and analyzed using the RCA amplicon kit (GE Healthcare). Genomic DNA (50 ng) was incubated for 3 min at 95 °C in sample buffer and placed on ice for 5 min. Enzyme mix and reaction buffer were added, and the samples were incubated at 30 °C for 18 h for amplification, followed by incubation at 65 °C for 15 min to inactivate the enzyme. NcoI was added to the samples and incubated for 1 h at 37 °C, and the digested samples were resolved on 1 % agarose gels.