Genome-wide gene expression profiling in 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 determine transcriptome changes 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 RNA extraction The tissues were collected 7 days after the injury, placed immediately in liquid nitrogen and stored at -80°C. Total RNA was extracted from 3 mm rings surrounding the initial punch wounds using RNeasy Mini kit (Qiagen). Each RNA sample was extracted from a pair of ear pinna rings from the same animal.  RNAseq RNA samples from a pair of ear pinnae from each animal were pooled. The quantity of the extracted RNA was determined fluorometrically on Qubit RNA assay (ThermoFisher Scientific), while the RIN (RNA integrity number) was determined using RNA Screen Tape Analysis on 4150 TapeStation System (Agilent). TruSeq Stranded Total RNA with Ribo-Zero Human/Mouse/Rat kit (Illumina) was used for library construction, followed by 5' and 3' adapter ligation. The cDNA libraries were sequenced in a paired-end mode on the Illumina platform as a service provided by Macrogen.  Data processing Sequencing data was converted into FASTQ format with Illumina's bcl2fastq converter. All samples were trimmed using Trim Galore (version 0.6.7), and these trimmed reads were successfully aligned to Gencode's GRCm39 Release M28 mouse reference genome using STAR (version 2.7.10a). Transcript assembly and estimation of the relative abundances were performed with HTSeq-count (version 2.0.2). Raw counts were then normalised using DESeq2 (version 1.36.0).