Sensifast probe mix

qPCR of pBBR1MCS-5 plasmid contained within OMVs was performed as previously described (11 (link)). Briefly, qPCRs consisted of 20 μM forward and reverse primers (forward primer, 5′-TTCGGAGACGTAGCCACCTA-3′; reverse primer, 5′-CAACAACCGCTTCTTGGTCG-3′), SensiFAST probe mix (Bioline, United Kingdom), and 2 μL of sample (control or DNase-treated OMVs), plasmid DNA as a positive control, or MilliQ H₂O as a negative control, and volumes were made up to 20 μL with MilliQ H₂O in a 96-well qPCR plate (Bio-Rad, USA). qPCR was performed using a CFX Connect real-time PCR system (Bio-Rad, USA) with cycle parameters consisting of denaturing at 95°C for 30 s and annealing at 55°C for 15 s. Gm^r gene copies as a measure of plasmid copy number were calculated as previously described (42 (link)), using the following equation: [(6.022 × 10²³ copies per mole) × (DNA amount in grams)]/[(DNA length in base pairs) × (660 grams per mole per base pair)]. Threshold cycle values were plotted against the log of their initial template copy numbers using a standard curve generated with defined concentrations of purified plasmid ranging from 1 × 10⁻⁷to 1.0 ng.

The promoter sequences of IL-6, and Alu as the reference gene, were obtained from the Ensembl genome browser (www.ensembl.org). The forward and reverse primers, and probes were designed using the MethPrimer software tool (http://www.urogene.org/methprimer/) and procured as PrimeTime^® Std qPCR Assay (Integrated DNA Technologies, USA). Sequence of primers and probes used in the MethyLight assay were:
The assay efficiency was determined prior to running the assay according to method described in a previous study (18 (link)).
For every sample, 25 ng bisulphite-converted DNA was added to 1x SensiFAST™ PROBE mix (Bioline, UK) and 1x PrimeTime^® Std qPCR Assay in a 10 mL polymerase chain reaction (PCR) using Bio-Rad CFX96™ Real-Time System (Bio-Rad, USA). IL-6 and Alu were assayed in the same 96-wells plate. PCR reaction condition was 95 °C for 10 min (polymerase activation), followed by 95 °C for 10 s (denaturation) and 60 °C for 30 s (annealing and extension) and the cycle was repeated for 60 cycles. The IL-6 methylation of each sample was assessed as $Cq\left[\frac{IL-6}{Alu}\right]=\frac{\left[CqIL=6\right]}{\left[CqAlu\right]}$ .

Gene abundances (DNA) and transcription abundance (RNA) were estimated using quantitative PCR (qPCR). A TaqMan qPCR assay was used to quantify the archaea and bacteria 16S rRNA gene using universal primer pairs (Table 3) and a FAM-labelled probe with SensiFast Probe mix (Bioline) in 25 μL reactions. SYBR green qPCR assay was used to quantify the functional genes for methane production (mcrA), methane oxidation (pmoA) and AOA archaea ammonia monooxygenase (amoA) using universal primers (Table 3) in 8 μL reactions using SensiFast SYBR No-ROX (Bioline). All qPCR reactions were carried out on a BioRad CFX96 RT System C1000TM Thermal Cycler. PCR conditions were as follows: initial denaturation (94°C, 2 min) followed by 40 (TaqMan/SYBR analysis) cycles of denaturation (94°C, 5 sec) and hybridisation-elongation (annealing temperature, 45 sec). A subsequent melting temperature curve of the amplicon was performed in the SYBR green qPCR assays.