Genomic tips 20 g

Pheromone trapping in North America was used to collect wild E-pheromone and Z-pheromone preferring males using Scentry Heliothis traps baited with synthetic E (“New York”) and Z (“Iowa”) lures (Scentry Biologicals, Billings, MO, USA). Traps were placed directly next to sweet corn fields and males were collected from each trap every 1–2 weeks and stored at −20 °C. Lures were replaced every 2 weeks. Trapping of >20 males from each E and Z trap was done at three sympatric sites between 2010 and 2012 (Supplementary Table 4). Tissues were moved from −20 °C within 3 months of collection to at −80 °C for long-term storage. DNA was isolated from both Pennsylvania sites by grinding frozen tissues and using the Qiagen DNeasy tissue protocol (Qiagen, Germantown, MD, USA) without vortexing preserve high molecular weight DNA. DNA isolation of samples from Bellona, NY was conducted with Qiagen genomic tips (20 G). All samples were treated with Qiagen RNase. DNA concentrations were quantified using Qubit prior to sequencing.

High-molecular-weight (HMW) gDNA was extracted from the legs of flash-frozen spiders using Genomic-tips 20/G (Qiagen) based on previous studies [9 (link)]. The specimens were gently and quickly homogenized using a BioMasher II homogenizer (Funakoshi) and mixed with 2 ml of Buffer G2 (Qiagen), including 200 µg ml⁻¹ RNase A and 50 µl proteinase K (20 mg ml⁻¹). After incubation at 50°C for 12 h on a shaker (300 r.p.m.), the mixed lysate was centrifuged at 5000g for 5 min at 4°C, and the aqueous phase was loaded onto a pre-equilibrated QIAGEN Genomic-tip 20/G by gravity flow and washed three times. The DNA was eluted with a high-salt buffer (Buffer QF) (Qiagen), desalted and concentrated using isopropanol precipitation and resuspended in 10 mM Tris–HCl (pH 8.5). The extracted gDNA was qualified using a TapeStation 2200 instrument with genomic DNA Screen Tape (Agilent Technologies) and quantified using a Qubit Broad Range dsDNA assay (Life Technologies). The purified gDNA was size-selected (greater than 10 kb) with a BluePippin with High Pass Plus Gel Cassette (Sage Science).

8-OHdG, a well-known marker of oxidative stress, was measured in treated MCF-10A cells using the EpiQuik 8-OHdG DNA Damage Quantification Direct Kit (Colorimetric). Briefly, genomic DNA was extracted (using Qiagen Genomic-tips 20/G, Genomic DNA buffer set, and proteinase k) from previously treated cells and stored at −20 °C for later use in measuring 8-OHdG expression. To measure 8-OHdG, DNA was bound to a 96-well flat-bottom plate followed by a wash and the addition of the capture antibody. After the second wash, the detection antibody and enhancer solution were added. The color-developing solution was added to measure at an absorbance of 450 nm using the Biotek Synergy HTX Multi-mode microplate reader.

All the fungal strains were grown in a glycerol yeast extract liquid medium (2% glycerol, 0.4% yeast extract, pH 7). Mycelia were mechanically broken using a mortar and pestle with glass beads (425–600 μm, Sigma). DNA was then extracted using a Qiagen Genomic-tips 20/G (Qiagen, GmbH) following the manufacturer’s recommendation. Fungal ribosomal internal transcribed spacer regions were amplified using BMBC-F and ITS4-R primers as described elsewhere (Thimmappa et al., 2023 ), and the PCR products were purified using Wizard^® SV Gel and PCR Clean-Up System (Promega, USA). Amplicons were sequenced, and reads were assembled using PHRAP v1.090518 (de la Bastide and McCombie, 2007 (link)) and visualized with Consed v27.0 (Gordon et al., 1998 (link)). Species were identified by BLASTN¹ searches against the NCBI non-redundant (nr) database. Multiple matches with the same taxon among the ten best hits were considered correctly identified. Matches with more than one taxon among the top ten were labeled ambiguous.

At 60–72 h post-infection, infected cells from four 25 cm² tissue culture flasks were harvested using a scraper, frozen at −80 °C, thawed at 37 °C, and centrifuged at 25,830× g for 40 min at 4 °C. Supernatants were discarded and pellets were washed with ice-cold PBS, sonicated for 2 × 30 s to dissolve the pellets and centrifuged at 1000× g for 10 min at 4 °C to remove cellular debris. Supernatants were then centrifuged at 25,830× g for 40 min at 4 °C and pellets were washed with ice-cold PBS. The three-step centrifugation process was repeated. Genomic DNA was extracted from pellets with Qiagen 20/G Genomic-tips and a Genomic DNA buffer set according to the “Preparation of Gram-Negative and some Gram-Positive Bacterial Samples” protocol in the QIAGEN Genomic DNA Handbook.
DNA extracts were confirmed by analysis with a Nanodrop One spectrophotometer and Qubit 3.0 fluorometer (both from Thermo Fisher Scientific, Waltham, MA, USA) to confirm the quality and quantity. Capillary electrophoresis was applied to assess DNA integrity.

All yeast strains were grown to greater than 1 × 10⁷ cells/mL in 30 mL minimal media (16 (link)) or YPD (Supplemental Table S1). Genomic DNA from each strain was extracted using Qiagen 20/G Genomic tips from ∼1.5 × 10⁹ cells using the manufacturer's protocol.
All genomic DNA was barcoded using Oxford-Nanopore's native barcoding genomic DNA kit (EXP-NBD104), adapters were added using the ligation sequencing kit (SQK-LSK109). The manufacturer's protocol (versions NBE_9065_v109_revB_23May2018 and NBE_9065_V109_revP_14Aug2019) was followed with the following exceptions: incubation times for enzymatic repair step were increased to 15 min. All Agencourt AMPure XP beads were incubated for 30 min at 37°C before elution. Adapter ligation time was increased to 10 min. Multiplexed libraries were loaded on MinION flowcells (FLO-MIN106D R9) and run on a MinION sequencer (MIN-101B).