RNA sequencing of non-stimulated (non-stim) or lipopolysaccharide (LPS)-stimulated (100 ng/mL, 1 h) wildtype (WT) and mt-tRNAala m.5019A>G mutant murine bone marrow-derived macrophages (BMDMs). Three biological replicates per condition
The experiment was designed to examine differentially expressed genes between wildtype BMDMs and BMDMs with a heteroplasmic pathogenic mitochondrial DNA mutation in the mitochondrial tRNA for alanine, which disrupts mitochondrial translation. Both non-stimulated basal state and LPS-stimulated state BMDM transcripts were examined. RNA isolation was carried using RNeasy® Plus kit (74136, Qiagen) following manufacturer's suggestions and eluted RNA was purified using RNA Clean & Concentrator Kits (Zymo Research). RNA-seq samples libraries were prepared by Cambridge Genomic Services (CGS) using TruSeq Stranded mRNA (Illumina) following the manufacturer's description. For the sequencing, the NextSeq 75 cycle high output kit (Illumina) was used and samples spiked in with 1% PhiX. The samples were run using NextSeq 500 sequencer (Illumina). Differential Gene Expression Analysis was done using the counted reads and the R package edgeR version 3.26.5 (R version 3.6.1) for the pairwise comparisons.