yunxia, G. | bioRxiv Science

bioRxiv2025

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 dependent on the quality of source material, and post-mortem interval (PMI) is one of the major sources of variation in RNA quality. 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{degrees}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 36h. 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.

molecular biology↗

bioRxiv2024

snCED-seq: High-fidelity cryogenic enzymatic dissociation of nuclei for single-nucleus RNA-seq of FFPE tissues

Profiling cellular heterogeneity in formalin-fixed paraffin-embedded (FFPE) tissues is key to characterizing clinical specimens for biomarkers, therapeutic targets, and drug responses. Recent advancements in single-nucleus RNA sequencing (snRNA-seq) techniques tailored for FFPE tissues have demonstrated their feasibility. However, isolation of high-quality nuclei from FFPE tissue with current methods remains challenging due to RNA cross-linking. We, therefore, proposed a novel strategy for the preparation of high-fidelity nuclei from FFPE samples, cryogenic enzymatic dissociation (CED) method, and performed snRandom-seq (snCED-seq) for polyformaldehyde (PFA)-fixed and FFPE brains to verify its applicability. The method is compatible with both PFA-based and FFPE brains or other organs with less hands-on time and lower reagent costs, and produced 10 times more nuclei than the homogenate method, without secondary degradation of RNA, and maximized the retention of RNA molecules within nuclei. snCED-seq shows 1.5-2 times gene and UMI numbers per nucleus, higher gene detection sensitivity and RNA coverage, and a minor rate of mitochondrial and ribosomal genes, compared with the nuclei from traditional method. The correlation gene expression of nucleus from the post-fixed and the frozen sample can be up to 94 %, and the gene expression of our nuclei was more abundant. Moreover, we applied snCED-seq to cellular heterogeneity study of the specimen on Alzheimers Disease (AD) to demonstrate a pilot application. Scarce Cajal Retzius cells in older mice were robustly detected in our data, and we successfully identified two subpopulations of disease-associated in astrocytes, microglia and oligodendrocytes, respectively. Meanwhile, we found that most cell types are affected at the transcriptional level by AD pathology, and there is a disease susceptibility gene set that affects these cell types similarly. Our method provides powerful nuclei for snRNA-seq studies for FFPE specimens, and even helps to reveal multi-omics information of clinical samples.

bioengineering↗