Genome-wide DNA methylation profiling of regenerating ear pinnae in BALB/c mice induced with pharmacological epigenetic therapy involving subcutaneous administration of zebularine and retinoic acid in alginate hydrogel
Aim The experiment aimed to analyse genome-wide DNA methylation re-patterning in regenerating ear pinnae in mice in response to zebularine and retinoic acid treatment.  Animals The experiments were conducted on 8 to 10-week-old females of the BALB/c mouse strain in the Tri-City Academic Laboratory Animal Centre. After the mice were anaesthetised with isoflurane, through-and-through holes of 2 mm diameter were made in the ear pinna centre using a scissor-style ear punch (Hammacher Solingen; LOT FTC-15/8670/1). Next, the animals were randomised into groups of six. Immediately after wounding, the mice were administered subcutaneous injections of alginated hydrogel formulations. Mice in one group received zebularine (48 mg in 200 µl of 2% alginate hydrogel) and retinoic acid (0.8 mg in 200 µl of 2% alginate hydrogel), mice in the control group received 400 alginate hydrogel carriers each. The protocol for animal experiments was approved by the Local Ethics Committee for Animal Experimentation in Bydgoszcz (approval no. 51/2020).  Tissue collection and genomic DNA extraction The tissues were collected 7 days after the injury, placed immediately in liquid nitrogen and stored at -80°C. Genomic DNA was extracted from 3 mm rings surrounding the initial punch wounds using DNeasy Blood and Tissue kit (Qiagen.)  Each DNA sample was extracted from a pair of ear pinna rings from the same animal.  BSseq DNA samples from a pair of ear pinnae from each animal were pooled. Extracted DNA was quantified spectrophotometrically on the Varioskan Lux (ThermoFisher Scientific), and DIN (DNA integrity number) was determined with Genomic DNA Screen Tape Analysis on 4510 TapeStation System (Agilent). All samples with DIN >7.0 were further processed. Reduced-representation bisulfite sequencing (RRBS) was performed by the Genomic Core Facility at the University of Alabama at Birmingham using the Ovation RRBS Methyl-Seq kit (Tecan Genomics), followed by DNA libraries sequencing on NextSeq 500 platform (Illumina).  Data processing Following Tecan's Analysis Guide (https://github.com/nugentechnologies/NuMetRRBS), Trim Galore (version 0.6.7) was used to remove residual adapter sequences from the reads. Diversity trimming and filtering with their trimming script were then applied to the trimmed FASTQ files.