Thermocycler

The primers used in amplifying long overlapping fragments of mitochondrial genome were relative to their conserved regions (Table 1) [25 (link)]. Long-range PCR was used to amplify the whole mt genome of O. trifurcata in four overlapping fragments with locations of amplicons between rrnS and cytb (~3 kb), cytb and cox1 (~4 kb), cox1 and rrnL (~3 kb) and rrnL and rrnS (~5 kb). The long PCRs were performed by making a total volume of 50 µl per amplicon, with the reaction mixture containing 34.75 µL dH₂O, 5 µL of 10× Thermopol reaction buffer (Biolabs, New England), 10 mM of each dNTP (Takara, Dalian, China), 1.25U LATaq (Takara, Dalian, China), 2 µM of each primers (TsingKe, Beijing, China) and 2 µL of genomic DNA in a thermocycler (Biometra, Göttingen, Germany). The PCR conditions for the amplification were initiated by denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation for 30 seconds at 94 °C, annealing for 30 seconds at 50 °C, extension for 5 min at 60 °C, with 7 min of final extension at 60 °C, and finally the reaction was stopped at 4 °C. The obtained amplicons were then cloned into pGEM-T-Easy vector (Promega, USA), which were sequenced (Sangon BioTech company, Shanghai, China) employing a strategy of primer-walking [26 (link)]. The complete mitochondrial genome of O. trifurcata (GenBank accession no. MK227249) was thus obtained.

Dynabeads^TM Oligo(dT) 25 (Invitrogen, Darmstadt, Germany) were used to isolate mRNA from single EB and TB cell samples. All isolated mRNA was further used for cDNA synthesis. All procedures were carried out according to the manufacturer’s instructions with previously described modifications by [74 (link)]. Whole mRNA samples were transcribed into cDNA using RevertAid^TM H Minus Reverse Transcriptase (200 U/µL) (Thermo Fisher Scientific, Dreieich, Germany) in a thermocycler (Biometra, Göttingen, Germany) as follows: 10 min at 25 °C, 1 h at 42 °C, and 10 min at 70 °C. Sterile water was added to the samples to a final volume of 100 µL.

Biomass from purified bacteria cultures on nutritive agar (Oxoid CM003) was transferred to tubes containing 10 mL of 0.3% saline sterile solution to achieve a 5 McFarland Standard cell suspension; then, 1 mL was transferred to microcentrifuge tubes (Microfuge 18 Beckman) and centrifuged for 5 min at 10,000 rpm.
The recovered cells were washed with the saline solution. For cell lyses, the suspensions were frozen at -20°C and then heated at 100°C for 10 min. The suspension obtained was immediately centrifuged. After 5 min at 10,000 rpm, 1 μL of lysed cell preparation was used in a PCR reaction with the Primer CsM13 [25 (link)]. The amplification was performed in a thermocycler (Biometra), according to Chambel et al. [26 (link)].
Visualization of PCR products was performed in a horizontal electrophoresis chamber (Pharmacia) with an Electrophoresis Power Supply EPS 600. DNA fragments were stained with 0.2 mg/mL of ethidium bromide and visualized with an ultraviolet transilluminator and Image system Alliance 4.7 Uvitec Cambridge. Data images were analyzed with the BioNumerics 6.6 software.

RNA was extracted from tissue preserved in RNAlater^® using RNeasy Kits from Qiagen (74104,74704) and treated with RNase-Free DNase (79254, Qiagen). To check for RNA quality, random samples were chosen and analyzed with Agilent RNA 6000 Pico Kit on a 2100 Bioanalyzer from Agilent Technologies, Waldbronn, Germany, according to manufacturer’s instructions. All samples had a RIN value from 9.40–10.0. cDNA was prepared with RevertAid H minus Reverse Transcriptase and Random hexamer primer (Thermo Fischer Scientific) in a ThermoCycler from Biometra. TaqMan assays were from ThermoFischer (Table 1). Gene expression was quantified by real time PCR using Maxima^™ Probe/ROX qPCR master mix (Thermo Scientific) and the 7900HT Fast Real-Time PCR System, Applied Biosystems, with the software SDS 2.4. Relative gene expression was calculated according to the 2^-ΔΔCT method with Rn18S as the housekeeping gene.

Total RNA was isolated from the spinal cord using an RNeasy Micro Kit (Qiagen). For complementary DNA (cDNA) synthesis, the reverse transcriptase reaction was performed using a Prime Script first-strand cDNA Synthesis Kit (Takara Bio). Quantitative RT-PCR was performed using primers specific to the genes of interest (Additional file 4: Table S1) and SYBR Premix Dimmer Eraser (Takara Bio). The data were normalized to glyceraldehyde-3-phosphate dehydrogenase. RT-PCR was conducted using a Thermocycler (Biometra, Göttingen, Germany) and products were detected by electrophoresis and ethidium bromide staining.