High-Molecular-Weight Spider gDNA Extraction

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).