Lightcycler 480ii

Total microbial DNA was extracted and purified using a QIAamp DNA Stool Kit (Qiagen, Hilden, Germany) and stored at -80°C. The 16S rRNA gene sequences of Bacteroidetes, Bifidobacterium spp., Clostridium cluster IV, Clostridium cluster XIVa, Escherichia coli, Firmicutes, Lactobacillus, and total bacteria (Raveh-Sadka et al., 2015 (link)) were cloned into the pMD19-T vector. Gene sequences were amplified from total colonic DNA using the primers listed in Table 3. A total of eight clones with 16S rRNA gene sequences belonging to different taxa were used as templates to test primer specificity. Standard curves were constructed with DNA from representative species for a concentration range from 10² to 10¹⁰ DNA copies/mL using a Lightcycler 480II instrument (Applied Biosystems). General microbial DNA extracted from colonic contents and specific DNA from recombinant microbiota were quantified using RT-PCR. The reaction conditions were as follows: 2 min at 50°C; an initial denaturation step at 95°C for 5 min; 40 cycles of denaturation at 94°C for 20 s, primer annealing at a species-specific temperature for 30 s, and primer extension at 60°C for 1 min (Decroos et al., 2006 (link)).

Trizol reagent (Solarbio, Beijing, China) was used to extract total RNA from MC3T3-E1 cells and bone tissue of SAMP6. Then TransScript II All-in-One First-Strand cDNA Synthesis SuperMix Kit (Crenscene, China) was used to synthesize the first-strand cDNA templates. According to the manufacturer’s protocol, RT-PCR was carried out by RT-PCR testing equipment (Roche LightCycler^® 480II, Switzerland), and the results were analyzed by Applied Biosystems 7500 Fast System SDS software. Using the expression levels of β-actin and calculated by the 2^−ΔΔCt method. The primer gene sequences used are listed in Table 1.

Total RNA was isolated from frozen adipose tissue using the RNA/DNA/Protein Purification Plus kit (Norgen Biotek Corp.). Briefly, approximately 100 mg of adipose tissue was cut, and the tissue was lysed in Buffer SKP (from the kit) using cold stainless-steel beads in a Tissuelyser (Qiagen) for 3 x 2 minutes at 25 Hz. RNA was then purified according to the protocol provided in the kit. cDNA was synthesized using 350 ng RNA input with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and diluted 1:2 in PCR-grade water. Real-time qPCR was performed using SYBR Green I Master (Roche) on a Lightcycler ^® 480 II (Applied Biosystems). Primers were purchased from Sigma (see Table S2). Gene expression was calculated relative to the expression of importin 8 (IPO8).

R. solanacearum strains were grown to an OD₆₀₀ of about 1.0 and total RNA were extracted by using a RNeasy Mini Kit (QIAGEN, Hilden, Germany). Contaminated genomic DNA was digested with DNaseI (Takara, Dalian, China) and confirmed by PCR using the primer pair for 16S rDNA. The cDNA was synthesized using the FastQuant cDNA first chain synthesis Kit (TIANGEN BIOTECH CO. LTD, Beijing, China) according to the manufacturer’s instructions. qRT-PCR was performed with Super Real PreMix Color SYBR Green (TIANGEN BIOTECH CO. LTD, Beijing, China) on Light Cycler480II (Applied Biosystems by Roche, Germany). The absolute value of –ΔΔCt = −(ΔCt1–ΔCt2) were calculated as described in the 2^-ΔΔCt method (Livak and Schmittgen, 2001 (link)). Each assay was repeated from RNA isolation for three independent experiments with at least three replications per trial. All the Ct data of rmy genes, phcA and phcR in EP1 and related mutants have been listed in Supplementary Tables S2-S4.

Total microbial DNA was extracted and purified using a QIAamp DNA Stool Kit (Qiagen, Hilden, Germany) and stored at −80°C. The 16S rRNA gene sequences of Bacteroidetes, Bifidobacterium spp., Escherichia coli, and Lactobacillus were cloned into the pMD19-T vector (21 (link)). Gene sequences were amplified from fecal total DNA using the primers listed in Table 2. A total of five clones with 16S rRNA gene sequences belonging to different taxa were used as templates to test primer specificity. Standard curves were constructed with DNA from representative species for a concentration range of 10²–10¹⁰ DNA copies/mL using a Lightcycler 480II instrument (Applied Biosystems, Carlsbad, CA, USA). The general microbial DNA extracted from feces and specific DNA from recombinant microbiota were quantified by RT-PCR. Reaction conditions were 2 min at 50°C, an initial denaturation step at 95°C for 5 min, and then 40 cycles of denaturation at 94°C for 20 s, primer annealing at a species-specific temperature for 30 s, and primer extension at 60°C for 1 min (22 (link)).

Total RNA was extracted from the cell lines using the NucleoSpin® RNA Plus kit (MACHEREY-NAGEL, #740984.250). cDNA was synthesized using a high-capacity cDNA reverse transcription kit (Applied BiosystemsTM, #4368814). GAPDH or β-actin was used as an internal control to quantify the mRNA levels of other genes. The sequences of gene-specific primers are listed in Supplementary Table 3. Relative mRNA levels were determined by RT-qPCR on a Light Cycler 480 II using the SYBR Green method (Applied BiosystemsTM, #4367659). Three biological replicates were used for each experiment. The relative mRNA expression levels were calculated using the 2 − ∆∆Ct method.

Genomic DNA was extracted from 200 μL of EMJH liquid clonal cultures. Extraction was performed using a commercial kit (QIAamp® DNA Mini Kit, Qiagen, Australia) according to the manufacturer's instructions. DNA elution was performed with 50 μL of buffer AE. The quantity of DNA was measured with NanoDrop™ (Thermo Fisher Scientific, Waltham, MA USA) and adjusted to 0.1 ng/μL.
Amplification of a fragment of the 16S rRNA gene was performed in a total volume of 10 μL on a LightCycler 480 II. The reaction mixture consisted in 2 μL PCR grade water, 5 μL of 2X reaction SYBR Green Mix (LC480 SYBR Green I Master kit, Roche, New Zealand), 0.5 μL of forward primer (5′-GGCGGCGCGTCTTAAACATG-3′), 0.5 μL of reverse primer (5′-CTTAACTGCTGCCTCCCGTA-3′), and 2 μL (0.2 ng) of DNA (Mérien et al., 1992 (link)). After amplification, the melting temperature of the amplified product was analyzed using LightCycler 480 Software.