Sybr green 1 dye

Total ribonucleic acid (RNA) was extracted from liver tissue using Trizol reagent (Beyotime Biotechnology, Shanghai, China). The total RNA was reverse transcribed into cDNA using reverse transcription kit from Takara Biomedical Technology, Japan. All primers were designed using Primer-BLAST on the National Center for Biotechnology Information (NCBI) website (Table 1). Using SYBR Green I Dye (Thermo fisher scientific, New York USA) according to the manufacturer’s instructions, Quantitative real-time RT-PCR (qRT-PCR) was performed using the lightcycler 480II real-time PCR system (Roche, Basel, Switzerland). The PCR cycling conditions were as follows: 95^°C for 5 min, 98^°C for 2min, followed by 40 cycles of 5 s at 98^°C, 5 s at 60^°C, 10 s at 95^°C, and a final step of 1 s at 65^°C. Gene expression analysis was performed by relative PCR amplification analysis (2–ΔΔCt).

LAMP reactions were prepared in single 0.2 mL PCR tubes in a laminar-flow hood. Each experiment was repeated independently three times. A 10× primer mix of LAMP primers consisting of 2 μM F3 and B3 primers, 16 μM FIP and BIP primers, and 4 μM FL and BL primers was prepared, aliquoted, and stored at −20 °C. Bst 3.0 reactions consisted of 2.5 μL 10× reaction buffer, 1.5 μL 100 mM MgSO₄, 3.5 μL 10 mM dNTPs, 2.5 μL 10× primer mix, 1 μL Bst 3.0 enzyme (NEB, #M0374S), and 2 μL 10 ng/µL DNA. Bst 2.0 Warm Start reactions consisted of 12.5 μL 2× Warm Start master mix (NEB #E1700S), 2.5 μL 10× primer mix, and 2 μL 10 ng/µL DNA. All reactions were assembled on ice. A no-template control was included to ensure amplification specificity. Reactions were incubated at either 65 °C for 30 min (Mth, Mco), 65 °C for 45 min (Mcu), or 58 °C for 45 min (Mbr) before the enzyme was heat inactivated at 80 °C for 5 min. Product detection was performed by carefully transferring 9 μL of the stopped reaction to a fresh 0.2 mL PCR tube and adding 1 μL of 1000× SYBR Green I dye (Thermo Fisher Scientific, Waltham, MA, USA). Visual inspection was performed with positive reactions turning yellow/green while negative reactions remained orange. DNA template amplification was additionally visualised by loading 5 μL of product on a 1.5% (w/v) agarose gel in 1× TAE following electrophoresis at 110 V for 45 min.

Total RNA was isolated from the cultured cells using TRIzol (Thermo Fisher) and then reverse-transcribed into cDNAs using a Universal cDNA Synthesis Kit (Exiqon, Vedbaek, Denmark). Real-time PCR was performed using the primer pairs for Mucin 16 (forward: 5′-ccccaaattccagaggtgaa-3′, reverse: 5′-tgacaaaggcgcactggtac-3′), MSLN (forward: 5′-cgccttgctttccagaacat-3′, reverse: 5′-attctgctgactgagcgcct-3′) and β-actin (ACTB, forward: 5′-agcgagcatcccccaaagtt-3′, reverse: 5′-gggcacgaaggctcatcatt-3′). SYBRGreen I dye (Thermo Fisher) served as a quantitative indicator in the qPCR reaction. An ABI PRISM 7900 Sequence Detection System [Applied Biosystems, Carlsbad, CA, USA) was used for PCR experiments. The cycle threshold (CT) of each qPCR assay was recorded, and the results were presented as 2^−ΔCT (ΔCT = CT(target) − CT(ACTB)].

To ensure only one copy of HRC was introduced into the deletion mutant genomes, we further determined the copy number of HRC in deletion mutants. Genomic DNA was extracted from each 4-day old 50 mL PDB culture using the DNeasy Plant Mini Kit (Qiagen, Inc.) following the manufacturer’s protocol. M-3125 served as the negative control, and strain ΔFv_08294 (known single HRC copy) served as the positive control. Quantitative PCR was performed using Platinum Taq DNA Polymerase (Thermo Fisher Scientific) and SYBR Green I dye (Thermo Fisher Scientific) following the manufacturer’s protocol, using the DNeasy extracted genomic DNA. The relative copy number of target genes was normalized to the single copy reference β-tubulin (FVEG_04081) gene (primers P1/50 and P1/51), and calculated via the 2^−ΔΔCt method [40 (link)].

Reverse transcription was performed on 1 µg total RNA using ImProm-II™ Reverse Transcription System (Promega, Madison, WI, USA) and random hexameric primers (Promega). Real time PCR reactions were performed with SYBR® Green I dye (Thermo Scientific, St. Leon-Rot, DE) on MyIQ Real Time thermocycler (Bio-RAD, Hercules, California, USA) according to manufacturer’s protocol and primers (Supplementary Table S1) (Eurogentec; Seraing, BE). Normalization was performed regarding to 36B4 gene level and the relative expression for a target gene was calculated using the comparative Ct method (2^−ΔΔCt).