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Single-nucleus RNA sequencing revealed the impact of post-mortem interval on the cellular component and gene expression analysis of mouse brains

ABSTRACT: Accurate analysis of cell atlas and gene expression in biological tissues using single-nucleus RNA sequencing (snRNA-seq) is closely related to the quality of their RNA quality, and post-mortem interval (PMI) is one of the major origins of RNA variation. Although the use of RNA-degraded tissues in transcriptome analysis remains controversial, such samples are sometimes the sole means to address specific questions. Current studies on the impact of PMI on transcriptome data are limited to large-scale RNA-seq, which ignores cellular heterogeneity. Thus, deciphering the non-cell-autonomous effects caused by PMI is imperative for understanding the cellular and molecular disruption it elicits. Here, we investigated the impact of PMI on cellular components and gene expression using snRNA-seq data from mouse brain tissues of post-mortem. We collected samples that were allowed to decay for varying amounts of time at 25 ̊C prior to snRNA-seq, covering the entire range of RIN values. The different effects on the PMI to the degradation rate of mRNA and rRNA within nuclei, and the mRNA presented a more stable state. Multi-channel analysis revealed the preferential transient depletion oligodendrocytes and OPCs with increasing PMI. In addition, a rapid widespread overregulation of ribosomal transient recruitment to protein (RP) genes in various cells, and reached a plateau at PMI of 36 h. Although state depletion of neuronal cells was not detected, we reported significant upregulation of PMI-dependent RP genes in its subpopulations and their cell loss. Moreover, RP genes showed the greatest differential expression in the subpopulations with greater cell perturbation, and we speculated that aberrant expression of these genes might be associated with cell death. In this study, we systematically investigated the changes in the transcriptome profile of brain tissue induced by PMI at single-cell resolution, and revealed one of the important factors that might be responsible for the changes. In addition, our data complemented a possible explanation for the changes in the cellular state of brain tissue induced by postmortem hypoxia-ischemia, and provided a reference for transcriptome studies of RNA degradation samples.

ORGANISM(S): Mus musculus

PROVIDER: GSE285936 | GEO | 2025/12/08

REPOSITORIES: GEO

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Project description:Recent developments in liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS)-based proteomics have drastically increased analytical depth and sample throughput, establishing a robust tool for comprehensive system-wide analyses. Such analyses are instrumental in studying the pathophysiology of diseases. One crucial source of material for this is post-mortem individuals. However, there is a significant gap in our understanding of the multi-organ temporal impacts of body decomposition on the proteome. This deficiency compromises result validity due to challenges in controlling the post-mortem interval (PMI). To address this, we reanalyzed published data to compare the effect of the PMI on the proteome alterations to the disease effects. Additionally, we conducted a clinically relevant time-course experiment using isogenic mice and applied LC-MS/MS in data-independent acquisition (DIA) mode to systematically study the proteome alterations post-mortem. Our experiment uncovered dramatic organ-specific protein regulation post-mortem, with only certain aspects attributable to protein autolysis suggesting active post-mortem protein regulation. Notably, the liver and spleen exhibited substantial proteome changes within hours of PMI, while the heart showed comparatively modest changes even after 96 hours. Subcellular fractionation of the heart led to an unexpected surge in the number of proteins exhibiting abundance differences early in PMI. We conducted a compressive analysis of semi-tryptic peptides for the first time, revealing distinct consensus motifs for different organs, indicating organ-specific protease activity. In conclusion, when designing LC-MS/MS-based studies, meticulous consideration of sample collection is crucial as PMI effects may outweigh disease effects. It is preferred that both experimental and control samples are taken in the operation room, especially for studies including subcellular fractionation or trans-organ comparison. In single-organ studies, the planning should involve careful control of the PMI, preferably by sample matching.

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Single-nucleus RNA sequencing revealed the impact of post-mortem interval on the cellular component and gene expression analysis of mouse brains

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