Dneasy blood and tissue extraction kit

DNA was extracted using the Qiagen DNeasy Blood and Tissue Extraction Kit™ (QIAGEN) and protocol. Extracted DNA was stained with SYBR Green™ (Thermo Fisher Scientific) dye, and agarose gel electrophoresis was performed to confirm the presence of high‐quality, high molecular weight genomic DNA and concentrations were quantified using a Nanodrop spectrophotometer (Thermo Fisher model # ND‐1000).

All animal experiments were approved by the Animal Ethics Committee of the QIMR Berghofer Medical Research Institute (project P1457) and ratified by the University of Queensland Animal Welfare Unit. Specimens of Rattus fuscipes were collected from the Department of Environment and Heritage Protection of the Queensland Government (permit WIS12109412). Specimens of Rattus fuscipes were trapped in Brisbane and surrounding regions using Eliot traps, baited with peanut butter and rolled oats. Rat faeces were directly examined by light microscopy for the presence of larvae consistent with Angiostrongylus sp. [3 (link)]; rats harbouring the parasite were euthanized with an overdose of CO₂ in a portable chamber for subsequent transport to the laboratory. Specimens of Angiostrongylus recovered from the pulmonary arteries of infected rats were identified to species morphologically [4 (link)] and washed extensively in physiological saline. Genomic DNA was isolated from amid-body section (to avoid ovaries) of an individual adult female worm using the QIAGEN DNeasy blood and tissue extraction kit, according to manufacturer’s instructions (Qiagen, Germany).

Genomic DNA was extracted from muscular tissues using the Qiagen^® DNeasy Blood and Tissue extraction kit (QIAGEN, Valencia, CA, USA) in accordance with the manufacturer’s protocol.
We generated GBS libraries by following the protocol of Elshire et al. [19 (link)]. Briefly, we digested genomic DNA at 37 °C with MseI restriction enzymes (New England Biolabs, NEB, Ipswich, MA, USA) and ligated an individual barcode to the cut sites. Then, we performed the PCR reaction, and PCR products were isolated to retain fragments of approximately 300–350 bp from an agarose gel using a Gel Extraction Kit (QIAGEN, Valencia, CA, USA). Next, the resulting library was sequenced on an Illumina NovaSeq6000 platform using the 150 bp paired-end protocol. These procedures were performed at Novogene Bioinformatics Technology Co., Ltd., Beijing, China (www.novogene.cn, 19 August 2019). Finally, clean reads were obtained by removing barcode adapters from raw Illumina data.

Anaplasma phagocytophilum-infected unfed ticks were microinjected with mock- or clock-dsRNA (~4.2 nl/tick). The microinjected ticks were incubated for 24 h for recovery. After 24 h, each group of microinjected ticks was fed on five uninfected mice. The engorged ticks were collected at 72, 96, 108, and 120 h post tick placement, and their tick body weight measurements were recorded immediately. Repleted ticks were cleaned with brushes to remove any mice hair or feces attached to the surface. Weight measurements were taken twice for each tick and average value was considered for the engorgement body weight analysis. In addition, the engorgement and feeding time for repleted ticks were recorded. After five days post-repletion of all engorged ticks, mice were euthanized and blood and tissues such as spleen and liver were collected separately from each group. Total RNA was extracted from the repleted ticks using Aurum Total RNA Mini kit (BioRad, Hercules, CA, USA). The total genomic DNA was extracted from murine tissues using the DNeasy blood and tissue extraction kit (Qiagen, Germantown, MD, USA). QRT-PCR analysis was performed to determine bacterial loads in murine blood and tissues. In addition, QRT-PCR was performed to analyze the expression of clock and immune genes in ticks

Host and parasite tissues were preserved and stored either frozen at -20°C or preserved in 70–100% ethanol at ambient temperature. Sections of worms, portions of tissues, or whole larvae in ethanol were incubated at room temperature in 1.5 mL microcentrifuge tubes for at least 12 hours to allow residual ethanol to evaporate. Frozen worm or tissue samples were thawed and portions removed for DNA extraction. All sample DNA extractions conducted at SCWDS for this study were carried out with the DNeasy Blood and Tissue Extraction Kit (Qiagen, Valencia, CA, USA) using the manufacturer’s protocol. DNA extracts were stored at -20°C.

DNA was extracted from DBS for parasite genetic diversity and population structure studies as previously described [26 (link)]. DNeasy Blood and Tissue extraction kit (Qiagen, Germany) was used to extract parasite DNA from DBS following the manufacturer's protocol.