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MULTI-seq: Universal sample multiplexing for single-cell RNA sequencing using lipid-tagged indices

ABSTRACT: We report the development of MULTI-seq: a scRNA-seq and snRNA-seq sample multiplexing approach using lipid- or cholesterol-modified oligonucleotides. We demonstrate MULTI-seq utility in the following scRNA-seq and snRNA-seq contexts: (1) Live HEKs and HMECs with and without stimulation with TGF-β, (2) Purified nuclei from HEKs, MEFs, and Jurkats stimulated for 0-24 hrs with ionomycin and PMA, (3) 96 unique HMEC cultures (with one technical replicate), and (4) human metastases and mouse immune cells isolated from cryopreserved primary tissue samples dissected from a patient-derived xenograft mouse model of triple-negative breast cancer at progressive stages of metastasis to the lung.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE129578 | GEO | 2019/04/11

REPOSITORIES: GEO

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Project description:The cardiac autonomic nervous system is crucial in controlling cardiac function, such as heart rate and cardiac contractility, and is divided into sympathetic and parasympathetic branches. Normally, there is a balance between these two branches to maintain homeostasis. However, cardiac disease states such as myocardial infarction, heart failure, and hypertension can induce the remodeling of cells involved in cardiac innervation, which is associated with an adverse clinical outcome. Although there are vast amounts of data for the histological structure and function of the cardiac autonomic nervous system, its molecular biological architecture in health and disease is still enigmatic in many aspects. Novel technologies such as single-cell RNA sequencing (scRNA-seq) hold promise for the genetic characterization of tissues at single-cell resolution. However, the relatively large size of neurons may impede the standardized use of these techniques. Here, this protocol exploits droplet-based single-nucleus RNA sequencing (snRNA-seq), a method to characterize the biological architecture of cardiac sympathetic neurons in health and disease. A stepwise approach is demonstrated to perform snRNA-seq of the bilateral superior cervical (SCG) and stellate ganglia (StG) dissected from adult mice. This method enables long-term sample preservation, maintaining an adequate RNA quality when samples from multiple individuals/experiments cannot be collected all at once within a short period of time. Barcoding the nuclei with hashtag oligos (HTOs) enables demultiplexing and the trace-back of distinct ganglionic samples post sequencing. Subsequent analyses revealed successful nuclei capture of neuronal, satellite glial, and endothelial cells of the sympathetic ganglia, as validated by snRNA-seq. In summary, this protocol provides a stepwise approach for snRNA-seq of sympathetic extrinsic cardiac ganglia, a method that has the potential for broader application in studies of the innervation of other organs and tissues.

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MULTI-seq: Universal sample multiplexing for single-cell RNA sequencing using lipid-tagged indices

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