High-Throughput RNA Sequencing of Fixed Tissues
Summary
Marking a pivotal advancement in M20 Seq technology's application to formalin-fixed paraffin-embedded (FFPE) samples, snRandom-seq emerges as a highly efficient snRNA-seq method, specifically tailored for FFPE specimens, and is characterized by its superior sensitivity and full-length attributes. The introduction of this novel technique proffers a resilient platform for single-cell analysis for both laboratory and clinical FFPE samples, significantly enhancing the detection of single-cell nucleus transcriptomes within FFPE specimens or other biologically inferior samples. It harbors immense potential for broader application in diverse biological research arenas and clinical methodologies, heralding a new epoch in the discipline.
Research Criteria
The primary objective of this article is to devise an innovative approach for conducting single nucleus total RNA sequencing in FFPE tissues. Through the utilization of random primers, the researchers successfully capture complete transcripts from individual nuclei within FFPE tissues, subsequently employing droplet-based barcoding and sequencing methodologies to generate extensive and highly sensitive snRNA-seq data. The authors comprehensively apply their groundbreaking technique to a diverse array of mouse and human FFPE samples, effectively showcasing its efficacy in facilitating cell type identification, RNA velocity analysis, and the discovery of rare subpopulations. Referred to as snRandom-seq, their methodology introduces a formidable tool for extracting valuable transcriptomic insights from FFPE tissues, with substantial implications for advancing biomedical research and enhancing clinical practices.
Fig.1 Experimental design. (Xu, 2023)
Sample Type
Mouse FFPE and fresh samples, cultured cell lines, and human FFPE samples.
Result—snRandom-seq has Discovered a Proliferative Subpopulation within the MTM Subtype of Liver Cancer FFPE Specimens
Applying snRandom-seq to a clinical FFPE sample of the MTM subtype of liver cancer, which had been preserved for about two years, yielded 5914 effective nuclei. The median gene count per nucleus was 3220, with 8182 UMIs. This process annotated the main cell types of the human liver and detected a wide range of RNA types in the sample, including lncRNAs, snRNAs, miscRNAs, miRNAs, and snoRNAs. Among them, a specific subgroup of liver cells (hepatocyte-2) highly expressed proliferation marker MKI67 and two other markers (ASPM and TOP2A), with most cells in the G2M phase. The communication between hepatocyte-2 and plasma cells involved specific ligand-receptor pairs, mainly receiving signals from plasma cells through the BMP signaling pathway. This has been reported to relate to liver cancer tumor progression. In summary, snRandom-seq discovered a specific proliferative and activated subgroup of liver cells from clinical FFPE specimens.
Fig.2 A proliferative subpopulation was found in the clinical FFPE human specimen by snRandom-seq. (Xu, 2023)
Result—snRandom-seq has Detected Different lncRNAs in Liver Cancer FFPE Specimens
Through the implementation of snRandom-seq on FFPE liver cancer samples, a substantial abundance of genes and unique molecular identifiers (UMIs) were successfully acquired, leading to the identification of principal liver cell clusters. Remarkably, prior investigations into single-cell transcriptomics have largely neglected the examination of long non-coding RNAs (lncRNAs). However, the application of snRandom-seq reveals a significant expression of lncRNAs within liver cell clusters, with distinctive lncRNA profiles observed across different clusters, implying potential divergences in disease mechanisms. With its inherent capacity to comprehensively cover full-length transcripts, snRandom-seq emerges as a promising avenue for elucidating the intricate landscape of lncRNAs in the realm of cancer biology.
Fig.3 snRandom-seq detected different lncRNAs in subpopulations of human normal HCC FFPE specimens. (Xu, 2023)
Creative Biolabs' Service
Single Cell Fixed RNA Sequencing Service
Creative Biolabs is proud to introduce our Single Cell Fixed RNA Profiling Service. Leveraging the innovative snRandom-seq method, we can now offer unparalleled insights into the transcriptomics of formalin-fixed paraffin-embedded samples. Dive deep into cell heterogeneity and unlock the potential of your FFPE samples with our cutting-edge service.
Learn moreCreative Biolabs offers single-cell Fixed RNA sequencing services based on snRandom-seq, an advanced technology that provides a granular view of the cellular landscape at the transcriptome level. It can capture the full-length information of the entire transcriptome of FFPE samples with high sensitivity through random primers, which will greatly promote the detection of single-cell nuclear transcriptomes in FFPE samples or other degraded biological samples. Compared with traditional single-cell technologies, snRandom-seq has a lower rate of doublets, a higher number of detectable genes, and uniform 5'-3' data coverage. Its coverage at the single-cell level and single-gene level is higher than the other two FFPE snRNA-seq methods.
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Reference
- Xu, Z.; et al. High-throughput single nucleus total RNA sequencing of formalin-fixed paraffin-embedded tissues by snRandom-seq. Nature Communications. 2023, 14(1): 2734.
