Optimized Nucleic Acid Extraction Protocol

DNA and RNA extraction methods have previously been described by Collard et al., 2022 [6 (link)]. Briefly, RNA and DNA were extracted by commercial kits using a Qiacube instrument (Cador Pathogen 96 QIAcube HT Kit, Qiagen France SAS, Courtaboeuf, France), and following the manufacturer’s recommendations with an additional bead-beating step to increase mechanical disruption. Freshly thawed 200 mg sample were mixed with ASL buffer at 4 °C and vigorously vortexed for 1 min. The suspension was transferred into a Pathogen Lysis Tube (Qiagen) containing two mg of sterile glass beads (100 μM diameter) and disrupted mechanically using a TissueLyser II (Qiagen GmbH, Hilden, Germany) for 10 min at 30 Hz. The suspension was then incubated at 95 °C for 5 min, vortexed for 15 s and centrifuged at 14,000 rpm for 1 min to eliminate any solid particles in subsequent steps. All samples were eluted in 150 μL AE buffer. Concentrations and purity of RNA and DNA were assessed by spectrophotometry (Nanodrop 2000 Spectrophotometer, Thermo Fisher Scientific, Waltham, MA, USA) via 260/280 and 260/230 absorbance ratios. Nucleic acids extracts were stored at −80 °C until further analyses.