Gene expression of chronically occluded tissue sections in terms of transcripts per million.

Gene expression of chronically occluded porcine femoral vein tissue sections in terms of transcripts per million. Here, two sections from three separate specimens were processed. Associated cell types based on deconvolution are also included.Histological regions of interest were identified and annotated on H&amp;E stained images. The corresponding tissue areas were dissected and processed for RNA isolation using the RNeasy FFPE kit (QIAGEN Cat#73504) according to the manufacturer instructions. Selected regions were deparaffinized, digested with Proteinase K and crosslinks were reversed before RNA purification. The resulting purified product was subsequently used for RNA sequencing. Strand-specific RNA-seq libraries were prepared using an TruSEQ total RNA-SEQ library protocol (Illumina provided). Library quality and quantity was assessed using an Agilent bio-analyzer and libraries were sequenced using an Illumina NovaSEQ-X2. The data were processed to count tables using the nf-core/rnaseq pipeline. Reads were trimmed using Trim Galore and then mapped to the Sscrofa11.1 genome assembly using STAR. Quantification of gene counts was done using Salmon. FastQC and MultiQC were used for data quality checking. The online version of CYBERSORTx was used for deconvolution. The safeTME matrix was exported from the spatialDecon R package and used as the signature matrix in CYBERSORTx. The raw count table was normalized by library size, scaled by gene length, and log2 transformed to be used as the mixture file in CYBERSORTx. The "Impute Cell Fractions" analysis module was run with the following parameters: Batch correction - disabled, Disable quantile normalization - true, Run mode (relative or absolute) - relative, Permutations - 1. The top 200 highly expressed genes in the RNAseq sample were overlapped with the safeTME signature genes to verify the deconvoluted major cell types.<br>