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Isolating peripheral blood mononuclear cells from HIV-infected patients for single-cell RNA sequencing and integration analysis.

ABSTRACT: Single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) allows the dissection of transcriptional changes in immune cells with HIV infection. Here, we isolate PBMCs from HIV-infected individuals. After counting the cell number and verifying cell viability, we perform scRNA-seq for PBMCs on the 10× Genomics protocol and the Illumina NovaSeq 6000 sequencing platform. Furthermore, we analyze the function and cellular trajectories of B cell subsets and B cell receptor (BCR) repertoire after filtering raw sequences data and normalizing gene expression. For complete details on the use and execution of this protocol, please refer to Jia et al. (2022).¹.

SUBMITTER: Jia J

PROVIDER: S-EPMC10140154 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature

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Isolating peripheral blood mononuclear cells from HIV-infected patients for single-cell RNA sequencing and integration analysis.

Jia Jie J Zhao Yu Y Wang Jian-Hua JH Kuang Yi-Qun YQ

STAR protocols 20230413 2

Single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) allows the dissection of transcriptional changes in immune cells with HIV infection. Here, we isolate PBMCs from HIV-infected individuals. After counting the cell number and verifying cell viability, we perform scRNA-seq for PBMCs on the 10× Genomics protocol and the Illumina NovaSeq 6000 sequencing platform. Furthermore, we analyze the function and cellular trajectories of B cell subsets and B cell receptor (BC ...[more]

PMID: 37060557

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Project description:BackgroundAcute myocardial infarction (AMI) can occur in patients with atherosclerotic disease, with or without plaque rupture. Previous studies have indicated a set of immune responses to plaque rupture. However, the specific circulating immune cell subsets that mediate inflammatory plaque rupture remain elusive.MethodsTen AMI patients were enrolled in our study (five with and five without plaque rupture; plaque characteristics were identified by optical coherence tomography). By single-cell RNA sequencing, we analyzed the transcriptomic profile of peripheral blood mononuclear cells.ResultsWe identified 27 cell clusters among 82,550 cells, including monocytes, T cells, NK cells, B cells, megakaryocytes, and CD34+ cells. Classical and non-classical monocytes constitute the major inflammatory cell types, and pro-inflammatory genes such as CCL5, TLR7, and CX3CR1 were significantly upregulated in patients with plaque rupture, while the neutrophil activation and degranulation genes FPR2, MMP9, and CLEC4D were significantly expressed in the intermediate monocytes derived from patients without plaque rupture. We also found that CD4+ effector T cells may contribute to plaque rupture by producing a range of cytokines and inflammatory-related chemokines, while CD8+ effector T cells express more effector molecules in patients without plaque rupture, such as GZMB, GNLY, and PRF1, which may contribute to the progress of plaque erosion. Additionally, NK and B cells played a significant role in activating inflammatory cells and promoting chemokine production in the plaque rupture. Cell-cell communication elaborated characteristics in signaling pathways dominated by inflammatory activation of classical monocytes in patients with plaque rupture.ConclusionsOur studies demonstrate that the circulating immune cells of patients with plaque rupture exhibit highly pro-inflammatory characteristics, while plaque erosion is mainly associated with intermediate monocyte amplification, neutrophil activation, and degranulation. These findings may provide novel targets for the precise treatment of patients with AMI.

Project description:Several previous studies have attempted to investigate the regulatory mechanisms underlying gene expression in ankylosing spondylitis (AS). However, the specific molecular pathways underlying this condition remain unclear. Previous research used next-generation RNA sequencing to identify a series of differentially expressed genes (DEGs) in peripheral blood mononuclear cells (PBMCs) when compared between patients with AS and healthy controls, thus implying that these DEGs may be related to AS. Furthermore, by screening these DEGS, it may be possible to facilitate clinical diagnosis and optimize treatment strategies. In order to test this hypothesis, we recruited 15 patients with AS and 15 healthy controls. We randomly selected five subjects from each group of patients for RNA sequencing analysis. Sequence reads were generated by an Illumina HiSeq2500 platform and mapped on to the human reference genome using HISAT2. We successfully identified 973 significant DEGs (p < 0.05) in PBMCs. When compared with controls, 644 of these genes were upregulated (with a fold?change?(FC) > 2) in AS patients and 329 were downregulated (FC < 0.5). Our analysis identified numerous genes related to immune response. Gene Ontology (GO) analysis indicated that these DEGs were significantly related to the positive regulation of epidermal growth factor-activated receptor activity, the positive regulation of the ERBB (erb-b2 receptor tyrosine kinase) signaling pathway, the differentiation of trophoblast giant cells, oxygen transport, immune-related pathways, and inflammation-related pathways. The DEGs were also closely related to the TNF and NF-?B signaling pathways. Six DEGs were verified by quantitative real-time polymerase chain reaction (qRT-PCR). Receiver operating characteristic (ROC) curve analysis indicated that IL6 may represent a useful biomarker for diagnosing AS. The development of new biomarkers may help us to elucidate the specific mechanisms involved in the development and progression of AS.

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Isolating peripheral blood mononuclear cells from HIV-infected patients for single-cell RNA sequencing and integration analysis.

Publications

Isolating peripheral blood mononuclear cells from HIV-infected patients for single-cell RNA sequencing and integration analysis.

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