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Scalable co-sequencing of RNA and DNA from individual nuclei.

ABSTRACT: The ideal technology for directly investigating the relationship between genotype and phenotype would analyze both RNA and DNA genome-wide and with single-cell resolution. However, existing tools lack the throughput required for comprehensive analysis of complex tumors and tissues. We introduce a highly scalable method for jointly profiling DNA and expression following nucleosome depletion (DEFND-seq). In DEFND-seq, nuclei are nucleosome-depleted, tagmented, and separated into individual droplets for mRNA and genomic DNA barcoding. Once nuclei have been depleted of nucleosomes, subsequent steps can be performed using the widely available 10x Genomics droplet microfluidic technology and commercial kits without experimental modification. We demonstrate the production of high-complexity mRNA and gDNA sequencing libraries from thousands of individual nuclei from both cell lines and archived surgical specimens for associating gene expression phenotypes with both copy number and single nucleotide variants.

SUBMITTER: Olsen TR

PROVIDER: S-EPMC9934633 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature

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Scalable co-sequencing of RNA and DNA from individual nuclei.

Olsen Timothy R TR Talla Pranay P Furnari Julia J Bruce Jeffrey N JN Canoll Peter P Zha Shan S Sims Peter A PA

bioRxiv : the preprint server for biology 20230210

The ideal technology for directly investigating the relationship between genotype and phenotype would analyze both RNA and DNA genome-wide and with single-cell resolution. However, existing tools lack the throughput required for comprehensive analysis of complex tumors and tissues. We introduce a highly scalable method for jointly profiling DNA and expression following nucleosome depletion (DEFND-seq). In DEFND-seq, nuclei are nucleosome-depleted, tagmented, and separated into individual droplet ...[more]

PMID: 36798358

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Project description:BackgroundLower extremity peripheral artery disease (PAD) is a growing epidemic with limited effective treatment options. Here, we provide a single-nuclei atlas of PAD limb muscle to facilitate a better understanding of the composition of cells and transcriptional differences that comprise the diseased limb muscle.MethodsWe obtained gastrocnemius muscle specimens from 20 patients with PAD and 12 non-PAD controls. Nuclei were isolated and single-nuclei RNA-sequencing was performed. The composition of nuclei was characterized by iterative clustering via principal component analysis, differential expression analysis, and the use of known marker genes. Bioinformatics analysis was performed to determine differences in gene expression between PAD and non-PAD nuclei, as well as subsequent analysis of intercellular signaling networks. Additional histological analyses of muscle specimens accompany the single-nuclei RNA-sequencing atlas.ResultsSingle-nuclei RNA-sequencing analysis indicated a fiber type shift with patients with PAD having fewer type I (slow/oxidative) and more type II (fast/glycolytic) myonuclei compared with non-PAD, which was confirmed using immunostaining of muscle specimens. Myonuclei from PAD displayed global upregulation of genes involved in stress response, autophagy, hypoxia, and atrophy. Subclustering of myonuclei also identified populations that were unique to PAD muscle characterized by metabolic dysregulation. PAD muscles also displayed unique transcriptional profiles and increased diversity of transcriptomes in muscle stem cells, regenerating myonuclei, and fibro-adipogenic progenitor cells. Analysis of intercellular communication networks revealed fibro-adipogenic progenitors as a major signaling hub in PAD muscle, as well as deficiencies in angiogenic and bone morphogenetic protein signaling which may contribute to poor limb function in PAD.ConclusionsThis reference single-nuclei RNA-sequencing atlas provides a comprehensive analysis of the cell composition, transcriptional signature, and intercellular communication pathways that are altered in the PAD condition.

Dataset Information

Scalable co-sequencing of RNA and DNA from individual nuclei.

Publications

Scalable co-sequencing of RNA and DNA from individual nuclei.

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