biostudies-arrayexpressDylan RyanMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15591The 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.biostudies-arrayexpressNucleic Acid Extraction - 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).Sequencing - 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).Sample Collection - BMDMs were plated at 0.5 x 106 cells/mL (2 mL total volume) in a 6-well dish and left to adhere overnight at 37oC in a de-humidified incubator (21% O2, 5% CO2) before being treated with LPS (100 ng/mL) or left untreated.Library Construction - RNA-seq samples libraries were prepared by Cambridge Genomic Services (CGS) using TruSeq Stranded mRNA (Illumina).OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - 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. EdgeR computes effective library sizes using TMM (i.e trimmed mean of M-values) normalization. The normalization factors account for sequencing depth and RNA composition. Correct for GC content and gene length bias for all genes which pass filtering. GC content bias affects the amplification of fragments, which in turn affects read coverage and could confound true signals so therefore needs to be corrected for. We used the CQN bioconductor package (version 1.30.0) which allows removal of a systematic GC content and gene length bias by a smoothing function. This should ensure that there is no bias towards GC rich reads, compared to those with low GC content. The same applies with gene lengths.We used a generalised linear model (glm) edgeR approach to make pairwise comparisons between groups. We then adjust for multiple testing via the FDR (Benjamini-Hochberg) approach used by edgeR.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsNextSeq 500RNA-seq of coding RNAMus musculusDylan RyanfalseRNA 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 conditionThe 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.2025-09-12T00:00:00Z2026-06-16T10:50:29.659Z2025-09-12T12:42:12.751ZE-MTAB-15591ERP180019EFO_0002944EFO_0004170EFO_0005518EFO_0003816EFO_0003738EFO_0004184