RNA sequencing of BM-MDSCs exposed to high potassium
Total RNA was extracted from BM-MDSCs treated with or without additional 34.7 mM KCl. Libraries were prepared according to standard Illumina protocols. RNA sequencing was performed by Shanghai Biotechnology Corporation (Shanghai, China). The raw reads were filtered by Seqtk before mapping to genome using Hisat2 (version:2.0.4). The fragments of genes were counted using stringtie (v1.3.3b) followed by TMM (trimmed mean of M values) normalization. Differential gene expression analysis was conducted using DESeq2 (version 1.20.0), with a significance threshold of a corrected p-value < 0.05 and an absolute fold change = 2. The distribution of differentially expressed genes in KEGG pathways was analyzed using the clusterProfiler R package (version 3.8.1) to identify key biological mechanisms. Overall design: BM cells were isolated from C57BL/6J mice and stimulated with 40 ng/mL recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF, Peprotech) for four days. Cells were cultured either in the absence or presence of additional 34.7 mM KCl (Sigma)