# Impact of Long-Term Cannabidiol (CBD) Treatment on Mouse Kidney Transcriptome
## 2 Material and Methods  
The experiment involved 12 male mice of the C57 BL/6J strain, starting at an age of 60 days. Throughout the experiment, the mice were kept at a temperature of 22 \({}^{\circ}\)C (\(\pm\)2 \({}^{\circ}\)C) and a humidity level of 55% (\(\pm\)10%), with a light cycle of 12 h of light and 12 h of darkness. Mice were provided with ad libitum access to food and water. This study included three groups of mice, each consisting of 4 individuals. The experimental groups received a daily intraperitoneal injection of a CBD solution (200 \(\mu\)L) for 28 days. The CBD was dissolved in saline with 2% Tween 80, and the groups differed in CBD dosage, receiving either 10 mg/kg or 20 mg/kg of body weight (b.w.). The control group received saline (200 \(\mu\)L) with 2% Tween 80. After the 28-day period, the mice were euthanized using inhalation anesthesia with isoflurane, followed by spinal cord transection. All animal procedures were reviewed and approved by the II Local Ethics Committee in Krakow (permission number 90/2022). After euthanasia, kidneys were collected for RNA isolation using Trizol reagent (Thermo Fisher, Waltham, MA, USA). The quality of the obtained RNA was assessed using TapeStaion 4150 (Agilent, Santa Clara, CA, USA) analysis and RNA integrity number evaluation (RIN \(>\) 8). The Poly(A) RNA Selection Kit (Lexogen, Wien, Austria) was used to purify RNA fragments containing polyadenine (poly(A)) tails. For library preparation, 50 ng of mRNA and the CORALL RNA-Seq V2 Library Prep Kit with UDIs (Lexogen, Wien, Austria) were used. The resulting libraries underwent quality assessment and were sequenced commercially with paired-end 2 \(\times\) 150 bp reads on a NovaSeq6000 system (Illumina, San Diego, CA, USA), generating datasets with a minimum of 28 million reads per sample. Quality control of raw reads was performed using FastQC software (v0.11.9). Sequence trimming and filtering were carried out using Flexbar software (3.5.0) [13]. The filtered reads were then mapped to the mouse GRCm39 reference genome using STAR software (2.7.5c) [14]. Read counts were obtained using Htseq-count software (1.99.2) [15]. Normalization and differential expression analyses were conducted using DESeq2 software [16] implemented in iDEP2.0 (integrated Differential Expression and Pathway analysis, v2.01) [17]. iDEP2.0 was also used for analyzing expression profile diversity through Principal Components Analysis (PCA) and hierarchical clustering based on Euclidean distance. Additionally, the software assessed the overrepresentation of differentially expressed genes in biological processes from the Gene Ontology (GO) database [18], as well as KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways, for visualization purposes only. [PERSON]'s exact test was applied for this purpose, using all known mouse genes as the background, and genes or processes with a corrected \(p\)-value (Benjamini-Hochberg correction) of <0.1 were considered significant. Raw reads, as well as raw read counts, were deposited in the Gene Expression Omnibus (GEO) and Sequence Read Archive (SRA) databases from the National Center for Biotechnology Information (NCBI) under the accession number GSE275998. The results of RNA-Seq were validated for four selected genes (_Ypel2_, _Dbp_, _Npas2_, and _Acmsd_) affected in at least one treatment using quantitative real-time PCR. The _Mrp132_ and _Ppi_ genes were used as an endogenous control [19; 20]. For cDNA synthesis, 500 ng of RNA was used with the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific), following the manufacturer's instructions. RT-qPCR was performed using the AmplIQ 5\(\times\) HOT EvaGreen(r) qPCR Mix Plus (ROX) kit (Novazym, Poznan, Poland) and primers designed to target mRNA sequences spanning two adjacent exons (sequences included in Supplementary File S1). Each sample was analyzed in triplicate on the QuantStudio 7 Flex system (Thermo Fisher Scientific). Relative gene expression levels were calculated using the \(\Delta\Delta\)Ct method.