Magmax express 96 deep well magnetic particle processor

Bacterial phylloplane colonies were randomly picked from two replicates per growth medium. Isolated colonies (n = 200, 40 per growth medium) were checked for purity by streaking, and grown for 24 h in their respective liquid growth medium at 30 °C on a shaker (150 rpm), washed and resuspended in 2 mL of sterile 10 mM MgSO4 solution to obtain suspensions containing bacteria in mid-exponential phase (OD_{600 nm} = 0.4). Next, 20 μL of this bacterial suspension was used for the detection of IAA production using the Salkowski’s reagent method [54 (link)], for the detection of acetoin production using the Voges–Proskauer test [55 (link)], and for assessing ACC deaminase activity by monitoring the amount of α-ketobutyrate generated by the enzymatic hydrolysis of ACC [56 (link)]. Genomic DNA of all 200 isolates was extracted using a MagMAX DNA Multi-Sample Kit (Life Technologies, Carlsbad, CA, USA) and a MagMAX Express-96 Deep Well Magnetic Particle Processor (Life Technologies, Carlsbad, CA, USA). The portion of the bacterial 16S rRNA gene was PCR-amplified using 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′) primers, and 20 μL of the PCR product was used for unidirectional Sanger sequencing using the 27F primer by Macrogen Europe (Amsterdam, The Netherlands).

As previously described14 (link)16 (link), RNA was extracted from gamma-irradiated-Rift Valley fever virus-spiked (RNA extraction-positive control) blood, oral, rectal and urine samples inactivated in lysis buffer solution using the MagMAX Pathogen RNA/DNA Kit (Life Technologies) with the MagMAX Express-96 Deep Well Magnetic Particle Processor (Life Technologies).
Reverse-transcribed MARV and Rift Valley fever virus RNA were detected on the ABI 7500 Real-Time PCR System (Life Sciences) using the SuperScript III Platinum One-Step Q-RT-PCR Kit (Life Technologies), with amplification primers and reporter probes targeting the viral protein 40 gene and the large segment, respectively (Supplementary Table 3). Relative MARV TCID₅₀eq ml⁻¹ were interpolated from standard curves generated from serial dilutions of the titrated MARV 371 bat strain spiked into appropriate biological specimens.

We extracted RNA (90 μL) from the 2013 (800 μL) and 2017 (500 μL) tick pool lysates using the MagMax Pathogen RNA/DNA Kit on the MagMax Express-96 Deep Well Magnetic Particle Processor (Thermo Fisher Scientific). KASV has an 18.3-kb single-stranded, negative-sense, trisegmented RNA genome comprising large segment that encodes for the viral RNA-dependent RNA polymerase (RdRp), medium segment that encodes for the glycoprotein precursor (GP), and small segment that encodes for the nucleoprotein (N) (2 (link)). We analyzed RNA by quantitative reverse transcription PCR (qRT-PCR) using the SuperScript III Platinum One-Step qRT-PCR Kit (Thermo Fisher Scientific) with primers and probes (Appendix Table 1) targeting the KASV N gene, tick mitochondrial 16S ribosomal RNA (rRNA) gene (16 (link)), and eukaryotic 18S rRNA gene (Thermo Fisher Scientific; 2017 tick pools only). Relative KASV RNA copies/tick pool were interpolated from a standard curve generated from a serial dilution of a known concentration of a synthetic KASV RNA oligo.

Two methods were employed to isolate RNA. (i) Cells were collected for RNA isolation from 2D controls by washing the cells once with DPBS^−/−, followed by scraping the cells into DPBS^−/−. The resulting cell suspension was pelleted by centrifugation at 300x g for 1 minute at room temperature. The supernatant was carefully removed, and TRIzol (Life Technologies) was added to lyse the cells. For organoids, RNA isolation was performed by removing 2 ml of suspension culture medium from the Erlenmeyer flasks and collecting the organoids by centrifugation at 300x g for 5 min at room temperature. The supernatant was carefully removed, and organoids were washed with 5 ml of DPBS^−/− and repelleted. DPBS^−/− was gently removed, and TRIzol was added to lyse the organoids. TRIzol samples were then either processed immediately for RNA isolation according to the manufacturer’s instructions or stored at −80 °C for subsequent processing. RNA was quantified using a NanoDrop ND-1000 Spectrophotometer (NanoDrop). For vitamin K-dependent enzyme analysis, total RNA was isolated using the MagMAX™-96 Total RNA Isolation Kit on a MagMAX™ Express-96 Deep Well Magnetic Particle Processor as described by the manufacturer (both from Thermo Fisher Scientific, Waltham, MA, USA).