4.8. RNA-Seq and Bioinformatic Analysis

Lungs were isolated from the WT and Cldn17^−/− animals and stored in Trizol^® before freeze-drying. RNA isolation was performed on the samples, and total RNA purity and concentration were assessed using spectrophotometry with the NanoDrop ND-1000 (ThermoFisher). RNA quality was evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), ensuring an RNA Integrity Number (RIN) greater than 5. Total RNA was processed for cDNA library preparation using the TruSeq Stranded Total RNA kit (Illumina, San Diego, CA, USA), which depletes cytoplasmic ribosomal RNA. Briefly, 800 ng of total RNA was treated with rRNA Removal Mix to deplete the rRNA. After purification, the RNA was fragmented into 200–300 bp segments and converted into cDNA fragments. A single ‘A’ base was added to the cDNA fragments, followed by ligation of the adapter. The products were purified and enriched through PCR to create the final cDNA library. The library was assessed for quality with the Bioanalyzer and quantified using Qubit (ThermoFisher). The libraries were pooled and sequenced on the NextSeq500 sequencing system using a 75-cycle paired-end protocol. BCL files generated by the NextSeq500 were converted to FASTQ files for downstream analysis. Reads that passed quality control were aligned to the reference genome using the STAR aligner. The resulting BAM files were imported into Cufflinks and Cuffdiff tools from the Tuxedo Suite for differential gene expression analysis, with log2 fold changes and q-values calculated for replicates. Genes with p-values <0.05 were considered for further bioinformatics analysis.

Analysis of gene distribution, such as principal component analysis (PCA), volcano plot, and heatmap analysis, was performed using R version 4.3.2 and SRplot. Detailed pathway analyses were performed on genes that were differentially expressed in the lungs of Cldn17^−/− compared to WT mice employing the STRING database [50] and SRplot [51]. The pathway analysis of these genes was conducted on multiple databases such as the Kyoto Encyclopedia of Genes and Genomes (KEGG), Wiki, and Reactome. The gene set enrichment analysis (GSEA) and GO databases were used to scrutinize the cellular components, biological processes, and molecular functions of identified proteins and signaling pathways. The STRING database was utilized to demonstrate the gene-gene interactions [50]. Tissue-wide expression data were collected from the GTEx portal (https://www.gtexportal.org/home/ (accessed on 8 March 2025)).