Hydractinia DNA Extraction and Purification

DNA was extracted from Hydractinia embryos and adult specimens (see Appendix Table S2) by proteinase‐K (200 μg) incubation at 55°C for 2 h in buffered SDS solution (NaCl 0.1 M, Tris 10 mM, EDTA 25 mM, SDS 0.5%). One volume of Phenol‐Chloroform (1:1, v/v) was used to separate the DNA from the proteins. The DNA was then precipitated by sodium acetate–ethanol (0.1 and 3 v, respectively), washed with 70% ethanol, and dissolved in nuclease‐free water (adapted from Sambrook & Russell, 2001 ). Following RNaseA (ThermoScientific #EN0531) and RNaseT1 (ThermoScientific #EN0541) treatment, the DNA was further purified using a standard column‐based purification protocol (Escobar & Hunt, 2017 (link)). The purified DNA was then assessed by UV–Vis spectrophotometer, Qubit dsDNA‐BR (ThermoScientific # Q32850) and Qubit RNA‐HS assay (ThermoScientific # Q32852). Only DNA solutions with undetectable levels of RNA by Qubit RNA‐HS assay were used.