Formalin-fixed and paraffin-embedded (FFPE) tissue archives are the largest repository of clinically annotated specimens, and FFPE-compatible single cell gene expression workflow had been developed and applied recently. However, for tissues where cells are hard to dissociate or brains with complex neuronal cells, nuclear transcriptomic profiling are desirable. Moreover, the effects of standard pathological practice on the transcriptome of samples obtained from such archived specimens was also largely anecdotal. Here, we performed RNA-seq of nuclei from hippocampal of mice that underwent freezing, paraformaldehyde (PFA) fixation, and paraffin embedding. Then, we comprehensively evaluated the parameters affecting mRNA quality, transcription patterns, functional level and cell states of nuclei, including PFA fixation time and storage time of FFPE tissues. The results showed that the transcriptome signatures of nuclei isolated from fresh PFA-fixed and fresh FFPE tissues were more similar to matched frozen samples. By contrast, the brain fixed for more than 3 days had prominent impacts on the sequencing data, such as the numbers and biotypes of gene, GC content and ratio of reads interval. Commensurately, prolonged fixation time will result in more differentially expressed genes, especially those enriched in spliceosome and synaptic related pathways, affecting the analysis of gene splicing and neuron cells. MuSiC deconvolution results revealed that PFA infiltrating brains for 3 days will destroy the real cell states, and the proportion of neuron, endothelial and oligodendrocytes diminished while that of microglia was reversed. Yet the effect of storage time on cell composition was more neglectable for FFPE samples. In addition, oligodendrocyte precursor cells were most affected in all fixed samples, and their destruction was independent of fixation time and preservation time. The comprehensive results highlighted that fixation time had much more influences on the nuclear transcriptomic profiles than FFPE retention time, and the cliff-like effects appeared to occur over a fixed period of 1-3 days, with no more differences from additional fixation durations.
Zero-mode waveguides have become important tools for the detection of single molecules. There are still, however, serious challenges because large molecules need to be packed into nano-holes. To circumvent this problem, we investigate and numerically simulate a novel planar sub-wavelength 3-dimension (3D) structure, which is named as near-field spot. It enables the detection of a single molecule in highly concentrated solutions. The near-field spot can produce evanescent waves at the dielectric/water interface, which exponentially decay as they travel away from the dielectric/water interface. These evanescent waves are keys for the detection of fluorescently tagged single molecules. A numerical simulation of the proposed device shows that the performance is comparable with a zero-mode waveguide. Additional degrees-of-freedom, however, can potentially supersede its performance.
The combination of single-cell RNA sequencing and microdissection techniques that preserves positional information has become a major tool for spatial transcriptome analyses. However, high costs and time requirements, especially for experiments at the single cell scale, make it challenging for this approach to meet the demand for increased throughput. Therefore, we proposed combinational DNA barcode (CDB)-seq as a medium-throughput, multiplexed approach combining Smart-3SEQ and CDB magnetic microbeads for transcriptome analyses of microdissected tissue samples. We conducted a comprehensive comparison of conditions for CDB microbead preparation and related factors and then applied CDB-seq to RNA extracts, fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) mouse brain tissue samples. CDB-seq transcriptomic profiles of tens of microdissected samples could be obtained in a simple, cost-effective way, providing a promising method for future spatial transcriptomics.
Cells are basic building blocks of life with vast heterogeneity. Nowadays, the rapid development of single‐cell multiomics (scMulti‐Omics) has facilitated comprehensive understanding of gene regulatory networks, cellular characteristics, and temporal dynamics. However, simultaneous analysis of transcriptome and proteome at single‐cell level still faces huge challenges due to their differences in molecular modalities. Recent technological advances in single‐cell manipulations, barcoding, and ultrasensitive instrument recently offer unprecedented opportunities for the co‐profiling of genes and proteins. In this review, multiple types of single‐cell isolation, lysis, and molecular separation technologies are first introduced. Second, various approaches for co‐measurement of transcriptome and proteome in single‐cells are summarized, with their advantages, limitations, and capacity for targeted or unbiased deep analysis. Then we highlight the cutting‐edge spatial multiomics methodologies that operate at the single‐cell or subcellular resolution level, providing a comprehensive understanding of cell function and heterogeneity within the tissue spatial environment. The emerging biomedical applications of multiomics are also discussed. Finally, the challenges and prospects of this field are proposed.
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