Diethylpyrocarbonate treated water

RNA was extracted from cells and skin tissues according to the instructions of the RNAiso reagent manufacturer (TAKARA, Tokyo, Japan). Briefly, for cells, samples were washed three times with PBS and then homogenized at room temperature for 5 min by adding 1 mL of RNAiso. For skin, 40 mg of tissue was cut into several pieces, diluted with 1 mL of RNAiso, and homogenized by sonication with 10 cycles of 40 s/60 s working/resting time. All samples were then centrifuged at 12,000× g at 4 °C for 5 min, and the supernatant was transferred to a new tube. After vortexing with 0.2 mL of chloroform (Samchun, Pyeongtaek, Republic of Korea), the tube was incubated at room temperature for 5 min and centrifuged at 4 °C and 12,000× g for 15 min. The supernatant was transferred to a new tube, and 0.5 mL of isopropanol (Duksan, Seoul, Republic of Korea) was added. The tube was shaken, incubated at room temperature for 10 min, and centrifuged at 4 °C and 12,000× g for 10 min. The supernatant was removed, 1 mL of 75% ethanol (Supelco, St. Louis, MO, USA) was added to the remaining pellet, the tube was shaken several times, and the pellet was washed by centrifugation at 7500× g for 5 min at 4 °C. The liquid was then thoroughly removed, the remaining pellet was air-dried, and an appropriate amount of diethylpyrocarbonate-treated water (Biosesang, Seongnam, Republic of Korea) was added.

Total RNA was extracted from the cells using the RNAiso reagent (Takara Bio Inc., Shiga, Japan) according to the manufacturer’s instructions. Briefly, cells (1 × 10⁶ cells/mL) were washed with cold PBS and lysed with RNAiso reagent. Chloroform (Samchun, Seoul, Republic of Korea) was added to the lysate, and the mixture was centrifuged at 12,000× g for 15 min at 4 °C to separate the aqueous and organic phases. The aqueous phase was then transferred to a fresh tube, and RNA was precipitated by adding isopropanol (Duksan, Seoul, Republic of Korea) for 10 min at room temperature (RT). The RNA was centrifugated at 12,000× g for 10 min at 4 °C to precipitate the pellet. Subsequently, the RNA was washed with 75% cold-ethanol (Sigma-Aldrich, St. Louis, MO, USA), and centrifuged at 7500× g for 5 min at 4 °C. The washed RNA pellet was air-dried until the pellet was slightly transparent and dissolved in diethyl pyrocarbonate treated water (Biosesang, Seongnam, Republic of Korea). RNA quality and quantity were determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Rockford, IL, USA).