Proteinase k

Homogenates from brains, hearts, livers, spleens and kidneys were used to isolate total DNA. RNase (final concentration 5 µg/µl; EURx, Poland) was added to 300 µl of the lysate and incubated at 37°C for 30 minutes. Next, thermal inactivation of RNase was performed for 10 minutes at 65°C. To samples obtained in this way, 400 µl of Tissue Cell Lysis Solution (Lucigen, USA) and 5 µl of Proteinase K (concentration 25 mg/ml; EURx, Poland) were added, and then incubated for 30 minutes at 65°C. Samples were cooled in ice for 5 minutes, then 300 µl MPC Protein Precipitation Reagent (Lucigen, USA) was added and centrifuged (8,000 x g, 10 minutes, 4°C). Five hundred µl of isopropanol (POCH, Poland) were added to the supernatant and incubated at -20°C for 24 hours. Then, the samples were centrifuged (9,600 x g, 20 minutes, 4°C), the supernatant was removed, and 700 µl of 70% ethanol (POCH, Poland) were added to the resulting colorless pellet. The samples were centrifuged (9,600 x g, 40 minutes, 4°C), the supernatant was removed, and 500 µl of 70% ethanol were added to the white pellet. The supernatant was removed, and the pellet was dried for 20 minutes under vacuum at 30°C. The pellet was suspended in 30 µl of nuclease free water (Roth, Germany) and incubated for 15 minutes at 37°C to dissolve. The obtained samples were stored at -20°C.

Approximately 100 mg of the dermis was homogenized in the TissueLyser II (Qiagen, Hilden, Germany) in PBS at 4 °C, then digested with proteinase K (EurX, Gdańsk, Poland) for 16 h at 55 °C. DNA was isolated using the Tissue DNA Purification Kit (EurX). DNA was also isolated from primary fibroblast cultures using the Sherlock AX isolation kit (A&A Biotechnology, Gdynia, Poland).
DNA (100 ng) was denatured in 0.4 M NaOH and 10 mM EDTA for 10 min at 100 °C, neutralized with 2 M ammonium acetate, applied to a nitrocellulose membrane on the Bio-Dot instrument (Bio-Rad, Hercules, CA, USA), and UV-crosslinked. Dot-blots were blocked and then incubated with anti-5mC (1:2000, Abcam, Cambridge, UK) anti-5hmC, anti-5fC, or anti-5CaC antibodies (1:10,000, Active-Motif, La Hulpe, Belgium). Incubation with an anti-ssDNA antibody served as a loading control (1:1000, Enzo, New York, NY, USA). Next, the blots were washed in TBST and incubated with an appropriate secondary antibody. Signals were detected using the enhanced chemiluminescent (Bio-Rad) and GeneGnome Chemiluminescence imaging systems (Syngene, Cambridge, UK). The quantitative analysis was performed using Image Studio™ Lite software (LI-COR, Lincoln, NE, USA).