Single-Cell RNA-Sequencing of Peripheral Blood Mononuclear Cells with ddSEQ
Peripheral blood mononuclear cells (PBMCs) are blood cells that are a critical part of the immune system used to fight off infection. However, due to the complexity of PBMCs, which contain multiple different cell types, studying the function of the indivi
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Introduction The immune system is complex, and its function involves interplay of a variety of cells, tissues, and secreted molecules such as cytokines and chemokines. It plays a crucial role in recognition and removal of foreign or “nonself” material, pathogens, cancer cells, and graft transplantations [1–4]. Failure of the immune system to recognize tissue or cells as “self” can lead to autoimmune diseases and, on the other hand, defects in immune system ability to fight foreign invaders results in immune deficiency and susceptibility to infections. Failure in regulating immune system responses results in myriad of inflammatory diseases. Harnessing the power of the immune system to fight against cancer, such as immune checkpoint therapy, has been successful against a variety of cancer types. To delineate the mechanisms that drive immune function in both health and disease states, researchers often perform gene expression studies on immune cells isolated from peripheral blood, including PBMCs, which include lymphocytes (B- and T-cells), dendritic cells, macrophages, and NK-cells [5]. RNA- Sequencing (RNA-Seq) provides an accurate method with which to measure gene expression of the
Valentina Proserpio (ed.), Single Cell Methods: Sequencing and Proteomics, Methods in Molecular Biology, vol. 1979, https://doi.org/10.1007/978-1-4939-9240-9_10, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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Shaheen Khan and Kelly A. Kaihara
whole transcriptome without prior knowledge of the genes expressed. However, traditional RNA-Seq is performed on cells processed in bulk, which averages gene expression and conceals the underlying heterogeneity [6]. Single-cell RNA-Seq of PBMCs can provide in-depth assessment of gene expression of individual immune cell types that can reveal novel immune cell populations, identify pathogenic subsets of immune cells and transcriptional modules driving the pathogenesis, and identify biomarkers of efficacy and response to therapy [7]. Furthermore, single-cell RNA-Seq of PBMCs would be a key assay in analyzing a longitudinal sample in a large cohort of human patients since it is more readily available than tumor or tissue biopsies. PBMCs can be further enriched for rare subsets of immune cells, such as dendritic cells and monocytes, by selection protocols or cell sorting to gain detailed analysis of the transcriptome. The Illumina® Bio-Rad Single-Cell Sequencing Solution combines Bio-Rad’s innovative Droplet Digital PCR (ddPCR) technology with Illumina next generation sequencing (NGS) library preparation, sequencing, and analysis (Fig. 1). This solution provides a comprehensive, userfriendly workflow for single-cell RNA-Seq that enables controlled experiments with multiple samples, treatment conditions, and time points. Built and supported in collaboration between technology leaders, the Illumina Bio-Rad Single-Cell Sequencing Solution enables transcriptome analysis of hundreds to thousands of single cells across a wide range of cell sizes in a single experiment. T
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