Fisherbrand bead mill 24 homogenizer

Mature female U. diversus were collected in Riverside, California, USA by T. Dugger. High molecular weight genomic DNA was extracted from a single whole individual using the Gentra PureGene tissue kit (Qiagen, Valencia, CA, USA). For RNA, cephalothorax and total silk gland tissue (a combination of all silk gland types attached to the spinnerets) were isolated from individual spiders, flash frozen in liquid nitrogen, and stored at − 80 °C. Tissues were homogenized in TRIzol reagent using a Fisherbrand bead mill 24 homogenizer (Fisher Scientific, Waltham, MA, USA). RNA was then purified using the PureLink RNA Mini kit with on-column DNAse treatment (Ambion, ThermoFisher Scientific, Wilmington, MA, USA). Because of the small size of U. diversus, for each RNA extraction, tissues of the same type were combined from four individuals prior to homogenization. Nucleic acid quantification was done using a Qubit Fluorometer (Thermo Fisher Scientific, Wilmington, MA, USA) and RNA integrity was assessed with a Bioanalyzer (Agilent Technologies, Santa Clara CA, USA).

For each sample, we performed extractions using several fecal pellets (up to 0.25 g total). We extracted total DNA using the QIAamp PowerFecal DNA Kit (MO BIO Laboratories, Qiagen Co.) following the manufacturer's instructions with the following alterations; prior to homogenization, we incubated fecal samples in the provided lysis solutions for 10 min at 70°C. Next, we disrupted the fecal material in a Fisherbrand Bead Mill 24 Homogenizer (Fisher Scientific) at 6 m/s for 1–2 min, until the fecal slurry was fully homogenized. At the elution step, we eluted with 55°C PCR‐grade water and incubated columns for two minutes prior to centrifugation. In addition to our samples, we extracted one “blank” water sample per extraction kit. Purified DNA extracts were preserved at −25°C prior to library preparation.

Spleen tissues were homogenized in 1 mL of lysis buffer per 100 mg of sample using copper beads and the Fisherbrand™ Bead Mill 24 homogenizer (Thermo Fisher Scientific, Waltham, MA, USA). Total RNA was extracted using the Total RNA Kit (Solarbio Science & Technology Co., Ltd., Beijing, China) and protocol. The concentration and purity of extracted RNA was checked using the NanoDrop™ 2000/2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) at absorbance 260/280 and consequently stored at −80 °C for downstream analysis.

We applied the CTAB DNA extraction protocol as detailed in Schenk et al. (2023 , their Appendix S1) with the amendments described here and detailed in Appendix S2. Twelve replicates of leaf tissue from each sample (N_{species/silica} = 4, N_{species/herbarium} = 4) were weighed according to the amount of material available, which was less for herbarium (~5.0 mg) than silica‐dried (8.0–10.0 mg) specimens. Dry material was ground in a Fisherbrand Bead Mill 24 Homogenizer (Thermo Fisher Scientific, Waltham, Massachusetts, USA) for 2–4 min, with the longer grinding times used as needed to assure that the leaf material was finely ground. After homogenization, replicated lysates rested at room temperature for 15–20 min with the tube caps loosened to reduce foam and maximize the usable liquid volume. Lysates from the 12 replicates per species were pooled and mixed by pipetting to standardize DNA concentrations of starting material per replicate, then 400 µL of the pooled homogenate was aliquoted to 12 1.5‐mL microcentrifuge tubes. Replicates were randomly assigned to one of the four incubation treatments (50°C, 55°C, 60°C, and 65°C) and three duration treatments (1 h, 2 h, and “overnight” [18–20 h]). The temperatures and durations were based on commonly applied values in the literature as assessed by Schenk et al. (2023 ).

For lipid analysis, the cerebral cortex was dissected, weighed, and flash-frozen. 20 µl methanol per mg tissue and an internal standard were added to each sample and homogenized with 1.4 mm ceramic beads (shaken at 2.6 m/s for 30 s) using a Fisherbrand bead mill 24 homogenizer (Thermo Fisher Scientific). After centrifugation (20 min at 4 °C, 14,000 × g), the supernatant was transferred to a fresh tube, incubated for at least 1 h at − 20 °C, followed by an additional 20 min centrifugation (4,000 × g, 4 °C).

Individual whole mosquitoes were homogenized in 200 μl of MagMAX Lysis/Binding Buffer that was diluted 1:2 in phosphate buffer saline for 45 s using a Fisherbrand Bead Mill 24 Homogenizer (Thermo Fisher Scientific, Waltham, MA). Nucleic acid was extracted using the MagMAX-96 Viral RNA Isolation Kit (which isolates both RNA and DNA) and the KingFisher Duo Prime Purification System programed with the MagMAX Pathogen Standard Volume software protocol as described by the manufacturer (Thermo Fisher Scientific, Waltham, MA) with the following exceptions: 80 μl of homogenate was extracted, magnetic beads were washed with 250 μl of wash solution, and the nucleic acid was eluted in 50 μl. Notably, we employed the same nucleic acid extraction method that is widely used by vector control agencies to test mosquitoes for the presence of arboviruses [25 (link)]. Alternatively, RNeasy Plus Mini Kits (Qiagen, Mississauga, Ontario, Canada) were used to extract nucleic acid from mosquitoes, as recommended by the manufacturer (Qiagen, Mississauga, Ontario, Canada). RNA and DNA concentration in the samples was measured using a NanoDrop 2000 Spectrophotometer (ThermoFisher Scientific, Waltham, MA), according to the manufacturer recommendations.