Mini beadbeater

Bacterial RNA was obtained utilizing the RNAsnap method (25 (link)). Briefly, 1ml of saturated culture was centrifuged and the cell pellet was stored at −80°C. The pellet was then suspended in 500 μl RNAsnap mix (95% formamide, 18 mM ethylenediaminetetraacetic acid, 0.025% sodium dodecyl sulphate and 1% B-mercaptoethanol), lysed using ∼200 μl of 0.1mm Zirconia Silica beads [Biospec 11079101Z] using a BioSpec mini bead beater [Biospec 1001] for 2 min in a deep well 96-well plate [Axygen P-DW-20-C]. The plate was centrifuged and the clarified lysate was cleaned up using a Zymo ZR-96 RNA Clean & Concentrator kit [Zymo R1080] per manufacturer's instructions. Bacterial DNA was obtained using our standard automated DNA extraction pipeline based on a scaled down version of the Qiagen MagAttract PowerMicrobiome DNA/RNA Kit [Qiagen 27500–4-EP], detailed fully in (26 (link)).

For one mL C. campestris extracts, we collected 100 mg of the stem tissue in microcentrifuge tubes from C. campestris growing on H1706 tomato plants. We used the BioSpec Mini-Beadbeater to grind the liquid nitrogen-frozen tissue with five 2.3-mm diameter BioSpec zirconia beads and 1.0-mm diameter BioSpec zirconia beads in the tubes for 1 min, and then mixed with 1-mL deionized water. To remove the plant tissue debris, we centrifuged extracts for 30 s at 5,000 r.c.f., and used only the supernatant for untreated extract injections. For heat-treated extracts, we heated at 95°C for 5 min. For pH treated extracts, we adjusted the pH to 9 by adding 0.1-M NaOH. For filtered extracts, we filtered untreated extracts through a VWR 0.2-µm sterile syringe filter. We injected different treated extracts into the first internode of tomato stems using a syringe equipped with a 0.8 mm × 38.1 mm MonoJect needle. Furthermore, we used 3K, 10K, 30K, and 100K Amicon Ultra Centrifugal Filter Devices to filter Cuscuta extracts. Then, we use flow through extracts to do injection on H9553 stems to test the size of Cuscuta signals.

Supragingival and subgingival plaque samples were each vortex mixed for 30 s and Amies transport swabs were immersed in 0.5 mL FAB and mixed to remove bacteria. A crude DNA extract was prepared from each sample by digestion with proteinase K (100 µg/mL) at 60 °C for 60 min, followed by boiling for 10 min. Further DNA purification was conducted using a bead beating technique where 150 µL of each sample was mixed with 200 µL phenol saturated with Tris–HCl (pH 8.0), 250 µL glass beads (0.1 mm) suspended in TE buffer and 200 µL lysis buffer. Samples were then placed in a BioSpec Mini-Beadbeater for 2 min at 2100 oscillations/min and DNA extracted with the AGOWA mag Mini DNA Isolation Kit (AGOWA, Berlin, Germany).

Collected pellets were resuspended in 200 µl of TE buffer, and 50 ul of 10% SDS and 0.25 g of glass beads were added. The cells were disrupted by two 1 min cycles in a Biospec Mini-BeadBeater (Biospec Products, US) with 1 min incubation on ice in between cycles. To fully lyse the cells, 0.5 ml of TRIzol (TRIzol™ Reagent, Invitrogen) was added, and samples were vortexed and incubated at 70 °C for 20 min. Afterwards, 100 µl of chloroform was added, and samples were vortexed for 15 s and incubated for 2 min at RT. Followed by a centrifugation step (12.500 × g, 15 min, 4 °C), the aqueous phase (~ 300 µl) was transferred to a clean Eppendorf tube with 2 volumes of 100% ethanol. RNA precipitation was performed with 1:1 (v/v) isopropanol extraction and 20 mg/ml of glycogen. Samples were vortexed for 10 s and incubated at RT for 10 min. After centrifugation (12.000 × g, 5 min, 4 °C), the supernatant was removed, and the RNA pellet was washed with 100 µl of 70% ice-cold ethanol. Next, samples were centrifuged at 12.000 × g for 5 min at 4 °C and ethanol was removed, and RNA pellets were air-dried for 5 min. RNA was eluted with 30 µl of nuclease-free milli-Q water, and the quality was assessed by gel electrophoresis. RNA samples were stored at − 80 °C prior to sequencing.

Hippocampus and liver tissue were homogenized using the BioSpec Mini-Beadbeater (Biospec Products, Bartlesville, OK, USA) and CCF-STTG1 cells were washed with cold PBS. RNA was prepared using Trizol (Invitrogen, Carlsbad, CA, USA) and RNA was reverse transcribed to cDNA using the QuantiTect Reverse Transcription Kit (Qiagen), according to the manufacturer’s instructions. Quantitative PCR (qPCR) was conducted, as previously described, on a CFX384 Thermal Cycler (Bio-Rad Laboratories) using SYBR Green PCR Select Master Mix (Applied Biosystems, Warrington, UK) [55 (link)]. Relative quantification of gene expression was accomplished by using the comparative Ct method. Data were normalized to the most stable reference genes (Actb, B2m, Hprt1, and Sdha (hippocampus) or Actb (liver)), which were analyzed and selected with geNorm 3.5 and StepOnePlus. Expression levels are indicated by fold change values and compared to the WT mice on the vehicle control. Details of the primers used are shown in Table 1.

DNA was extracted from turtle tissues containing spirorchiid ova using the DNeasy Blood and Tissue kit (Qiagen, Hilden, GER). Procedure was as per the manufacturer’s instructions, except that the samples were homogenised using 1 g of 0.5 mm Zirconia/Silica beads (Daintree Scientific, Tasmania, Australia) in a Biospec Mini-Beadbeater for 3 mins followed by the incubation step. AL buffer (200 μl) was then added to the supernatant and extraction continued as per DNeasy kit instructions. DNA was eluted in 100 μL buffer AE.