4.11. RNA Sequencing of Wounds

RNA was isolated from hASC_exo- and hASC_exo-G-M-treated wounds on day 7 from 2 male and 2 female rats. Samples from each sex were pooled together for sequencing. The Qubit (Thermo Fisher, Waltham, MA, USA) and Agilent Tape Station (Agilent, Santa Clara, CA, USA) were used for measuring RNA concentration and quality. RIN was insured to be >8.0 for samples. The library was prepared using the TruSeq stranded mRNA Library Prep Kit according to the manufacturer’s instructions (Illumina, San Diego, CA, USA, Cat #: 20040532). The concentration and quality of the resulting DNA library were checked using the Qubit and Agilent Tape Station. Samples were loaded into the Illumina NextSeq 500 with 75 bp pair-end reads with indices. The NextSeq System Suite was utilized for real-time image analysis and base calling. All samples had a minimum of 40 million reads and sequences aligned to >80% to reference genome. Trimmomatic was used to trim reads and then a quality check was performed using FASTQC (v 0.12.1). Reads were mapped using HISAT2 (v 2.2.1) to rat genome GRCm39 (file downloaded from NCBI). Files were converted using SAMtools (v 1.21) and Feature-Counts (v 2.20.0) were used to determine reads. RStudio (v 2023.09.0+463) was used for analysis of differentially expressed genes (DEGs) with R package DESeq2 (v 1.42.1) and over-representation analysis (ORA) and gene set enrichment analysis (GSEA) gene ontology (GO) pathways were investigated using R package clusterProfiler (v 4.10.1).