C1000 touch deep well thermal cycler

The QX100^™ Droplet Digital^™ PCR System (Bio-Rad, Hercules, CA, USA) was used in this study according to the manufacturer’s instructions. Briefly, the ddPCR reaction mixtures (20 μL) contained: 1× ddPCR Supermix (Bio-Rad, Pleasanton, CA, USA), 750 nM of each primer, 750 nM of probe, 2 μL of Xcc sample. Droplets were generated using a Droplet Generator (DG) with an 8-channel DG8 cartridge and cartridge holder with 70 μL of DG oil/well, 20 μL of fluorescent PCR reaction mixture and a DG8 gasket. The prepared droplets were transferred to corresponding wells of a 96-well PCR plate (Eppendorf, Germany) using a multichannel pipette (Rainin, USA) by aspirating 40 μL from the DG8 cartridge. The PCR plate was subsequently heat-sealed with pierceable foil using a PX1^™ PCR plate sealer (Bio-Rad) and then amplified in a C1000 Touch^™ deep-well thermal cycler (Bio-Rad). The thermocycling protocol was: initial denaturation at 95°C for 5 min, then 45 cycles of denaturation at 95°C for 30 s, annealing at 54°C for 45 s (temperature ramp 2°C/s) and, finally, incubation at 98°C for 10 min and storage at 4°C. After cycling, the 96-well plate was fixed into a plate holder and placed into the Droplet Reader (Bio-Rad). Droplets of each sample were analyzed sequentially and fluorescent signals of each droplet were measured individually by a detector.

All gene-specific primers were designed by the NCBI online Primer-BLAST tools (www.ncbi.nlm.nih.gov/tools/primer-blast/ ) (Table S1, S2). Real-time quantitative PCR (qRT-PCR) analysis was carried out on a C1000 Touch™ deep-well thermal cycler (Bio-Rad, Hercules, CA, USA). One microgram of cDNA derived from mRNA and miRNA was used for qRT−PCR analysis according to the Quant qRT−PCR (SYBR Green) and miRcute miRNA (SYBR Green) Kit manuals. The mRNA and miRNA qRT−PCR reaction system was performed as follows: 1 µg of diluted cDNA was added as a template to each reaction system, which contained 1 µL of each reverse and forward gene-specific primer (10 µM), 10 µL of 2× TransStart Green qPCR Super Mix, and ddH₂O to bring the total volume to 20 µL. The 2^-ΔΔCT method was used to calculate the expression level of each gene with reference genes Actin and U6 (Table S1 and S2) (Livak and Schmittgen, 2001 (link)). Three biological replicates were performed for each gene.

Primers and genomic DNA were the same as those used for qPCR. The ddPCR assay was performed on the QX200™ droplet digital™ PCR system (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Briefly, 20 μL of ddPCR mixtures containing 1× ddPCR supermix (Bio-Rad, Hercules, CA, USA, Cat. No. 1863010), 800 nmol of each primer, 400 nmol of probe, and 1 μL of template were used to generate droplets. The emulsified samples were then transferred to PCR plates and amplified in a C1000 Touch™ deep-well thermal cycler (Bio-Rad, Hercules, CA, USA). After amplification, the droplets of each sample were analyzed in the QX200 droplet reader (Bio-Rad, Hercules, CA, USA), and the concentrations were analyzed using QuantaSoft software (version 1.7.4, Bio-Rad, Hercules, CA, USA). The T-DNA copy number was determined by comparing the concentration of P35S, NOS, or HPT II elements with the concentration of the CruA gene. Four technical replicates were performed per reaction.