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

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. Overall design: Eight-week-old male C57BL/6J mice were purchased from the Qing Long Shan Dong Wu Fan Zhi Chang, Nanjing, China The animals were anesthetized with 500 mg/kg tribromoethanol (Sigma, Saint Louis, MO, USA) and were killed by cervical dislocation. After the animals were sacrificed, brain tissues (hippocampus) were isolated, placed in 2 mL enzyme-free centrifuge tubes, and stored at 25 °C at constant temperature. Postmortem tissues were sampled at 0 h (snap frozen, H0), 24 h (H24), 36 h (H36), 48 h (H48), and 54 h (H54), followed by snap frozen in liquid nitrogen and stored at -80 °C until use. For the reliability of the data, we maintained the consistency of the environment for each sample and set up biological replicates of three mice per group.