An optimized protocol for single nuclei isolation from clinical biopsies for RNA-seq

Scientific Reports, Journal Year: 2022, Volume and Issue: 12(1)

Published: June 14, 2022

Abstract Single nuclei RNA sequencing (snRNA-seq) has evolved as a powerful tool to study complex human diseases. cell resolution enables the of novel types, biological processes, trajectories, and cell–cell signaling pathways. snRNA-seq largely relies on dissociation intact from tissues. However, tissues using small core biopsies presents many technical challenges. Here, an optimized protocol for single isolation is presented frozen later preserved kidney biopsies. The described fast, low cost, time effective due elimination sorting ultra-centrifugation. Samples can be processed in 90 min or less. This method obtaining normal morphology without signs structural damage. Using snRNA-seq, 16 distinct clusters were recovered peri-transplant acute injury allograft samples, including immune clusters. Quality control measurements demonstrated that these optimizations eliminated cellular debris allowed high yield high-quality library preparation sequencing. Cellular disassociation did not induce stress responses, which recapitulated transcriptional patterns associated with standardized methods isolation. Future applications this will allow thorough investigations biobank biopsies, identifying cell-specific pathways driving discovery diagnostics therapeutic targets.

Language: Английский

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Epigenetic signals that direct cell type–specific interferon beta response in mouse cells

Life Science Alliance, Journal Year: 2023, Volume and Issue: 6(4), P. e202201823 - e202201823

Published: Feb. 2, 2023

The antiviral response induced by type I interferon (IFN) via the JAK-STAT signaling cascade activates hundreds of IFN-stimulated genes (ISGs) across human and mouse tissues but varies between cell types. However, links underlying epigenetic features ISG profile are not well understood. We mapped ISGs, binding sites STAT1 STAT2 transcription factors, chromatin accessibility, histone H3 lysine modification acetylation (ac) mono-/tri-methylation (me1, me3) in embryonic stem cells fibroblasts before after IFNβ treatment. A large fraction ISGs STAT-binding was specific with promoter a STAT1/2 complex being key driver ISGs. Furthermore, to putative enhancers as inferred from co-accessibility analysis. dependent on context positively correlated preexisting H3K4me1 H3K27ac marks an open state, whereas presence H3K27me3 had inhibitory effect. Thus, present stimulation represent additional regulatory layer for type-specific response.

Language: Английский

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Optimized methods for scRNA-seq and snRNA-seq of skeletal muscle stored in nucleic acid stabilizing preservative

Communications Biology, Journal Year: 2025, Volume and Issue: 8(1)

Published: Jan. 4, 2025

Abstract Single cell studies have transformed our understanding of cellular heterogeneity in disease but the need for fresh starting material can be an obstacle, especially context international multicenter and archived tissue. We developed a protocol to obtain high-quality cells nuclei from dissected human skeletal muscle preservative Allprotect® Tissue Reagent. After fluorescent imaging microscopy confirmed intact nuclei, we performed four variations that compared sequencing metrics between enriched by either filtering or flow cytometry sorting. Cells (either sorted filtered) produced statistically identical transcriptional profiles recapitulated 8 types present muscle. Flow sorting successfully higher-quality resulted overall decrease input material. Our provides important resource obtaining single genomic tissue streamline global collaborative efforts.

Language: Английский

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Superloaded Multiplexed scRNA- s eq Data Preserves Primary Immune Cell Heterogeneity but Necessitates Stringent Doublet Removal

Immunological Investigations, Journal Year: 2025, Volume and Issue: unknown, P. 1 - 17

Published: Jan. 30, 2025

Single-cell RNA sequencing (scRNA-seq) has improved our ability to characterize rare cell populations. In practice, cells from different tissues or donors are simultaneously loaded onto the instrument (multiplexed) at recommended (standard loading) higher (superloading) numbers save time and money. Although cost-effective, superloading can stymie computational analyses owing high multiplet rates sample complexity. We compared effects of on multiplexed single-cell gene expression T receptor (TCR) data generated human thymus blood samples donors. Minimal transcriptomic differences were observed between by either standard superloading. Irrespective loading number, we found that over 50% expressing multiple TCR chains doublets. Multiple be run without compromising quality subsequent analyses. However, an additional doublet removal step based configuration may improve accuracy analysis.

Language: Английский

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Single-cell network biology enabling cell-type-resolved disease genetics

Genomics & Informatics, Journal Year: 2025, Volume and Issue: 23(1)

Published: March 27, 2025

Abstract Gene network models provide a foundation for graph theory approaches, aiding in the novel discovery of drug targets, disease genes, and genetic mechanisms various biological functions. Disease genetics must be interpreted within cellular context disease-associated cell types, which cannot achieved with datasets consisting solely organism-level samples. Single-cell RNA sequencing (scRNA-seq) technology allows computational distinction states provides unique opportunity to understand biology that drives processes. Importantly, abundance samples their transcriptome-wide profile modeling systemic cell-type-specific gene networks (CGNs), offering insights into gene-cell-disease relationships. In this review, we present reference-based de novo inference functional interaction have recently developed using scRNA-seq datasets. We also introduce compendium CGNs as useful resource cell-type-resolved genetics. By leveraging these advances, envision single-cell key approach mapping axis.

Language: Английский

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