Genomic DNA Extraction from Animal Tissues

Genomic DNA was isolated from the tissues of five animals in each group in ascending order of animal ID as described previously [76 (link)]. Briefly, frozen tissue was homogenized using a pestle in Dounce buffer and the homogenized tissue was transferred to an ice-cold centrifuge tube containing 0.5 mol/L sucrose. After centrifugation at 1,750 × g for 10 min, the supernatant was removed. Precipitated nuclei/cells were resuspended in 3 mL of Dounce buffer containing 0.002% RNase (NIPPON GENE Co., Ltd.), mixed with 3 mL of 0.2% proteinase K solution (FUJIFILM Wako Pure Chemical Co., Ltd.), and incubated at 50 °C for 2 h. The suspension was mixed with an equal volume of phenol/chloroform (1:1), rotated for 10 min, and centrifuged at 1,220 × g for 10 min. The aqueous layer was collected and extracted twice with phenol: chloroform. The aqueous layer was mixed with an equal amount of chloroform: isoamyl alcohol (24:1) and extracted in the same manner. Genomic DNA was precipitated by adding ethanol to the aqueous layer. The precipitated DNA was rinsed with 70% ethanol for 10 min, placed at room temperature, air-dried overnight, and dissolved in TE buffer (NIPPON GENE Co., Ltd.). The purified DNA was stored in a refrigerator NanoDrop (AGC TECHNO GLASS Co., Ltd.) was used to determine the DNA concentration.