Determination of DNA content

DNA extraction was performed using the salting-out method. Briefly, about 5 mg of lyophilized nerve tissue was placed in a sterile microtube and minced. The tissue was pre-treated with proteinase K (Cat# 19131, QIAGEN, Germany) for 16 h at 56 °C. Then, 0.5 volumes of saturated NaCl (Cat# S9888, Sigma Aldrich, USA) were added to the lysed tissues. The sample was vigorously shaken and then centrifuged at 13,000 rpm for 10 min at 4 °C. The supernatant was transferred to a new tube and an equal volume of isopropanol (Cat# 19516, Sigma Aldrich, USA) was added and mixed. The mixed samples were incubated at RT for 30 min. Centrifugation was performed at 13,000 rpm for 10 min at RT to isolate DNA. After discarding the supernatant from the tube, the DNA pellet was air-dried for approximately 5 min. Next, the dried DNA was dissolved in UltraPure distilled water (Cat# 10977-015, Invitrogen, USA). To quantify dsDNA using a fluorescent reagent that binds directly to it, we analyzed the extracted DNA with the Qubit dsDNA HS assay kit (Cat# Q32854, Thermo Fisher Scientific, USA) following the manufacturer’s protocol. The Qubit Flex fluorometer (Cat# Q33327, Thermo Fisher Scientific, USA) was used to detect fluorescence signals and calculate the concentration of dsDNA. The total amount of dsDNA was normalized to the dry weight of the tissue. dsDNA was quantified from three different parts of nerve tissue for each group.