scholarly journals No detectable alloreactive transcriptional responses during donor-multiplexed single-cell RNA sequencing of peripheral blood mononuclear cells

2020 ◽  
Author(s):  
Christopher S. McGinnis ◽  
David A. Siegel ◽  
Guorui Xie ◽  
Mars Stone ◽  
Zev J. Gartner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Rna Sequencing ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Transcriptional Responses ◽  
Peripheral Blood Mononuclear ◽  
Single Cell Rna Sequencing ◽  
Blood Mononuclear Cells

ABSTRACTSingle-cell RNA sequencing (scRNA-seq) provides high-dimensional measurement of transcript counts in individual cells. However, high assay costs limit the study of large numbers of samples. Sample multiplexing technologies such as antibody hashing and MULTI-seq use sample-specific sequence tags to enable individual samples (e.g., different patients) to be sequenced in a pooled format before downstream computational demultiplexing. Critically, no study to date has evaluated whether the mixing of samples from different donors in this manner results in significant changes in gene expression resulting from alloreactivity (i.e., response to non-self immune antigens). The ability to demonstrate minimal to no alloreactivity is crucial to avoid confounded data analyses, particularly for cross-sectional studies evaluating changes in immunologic gene signatures,. Here, we compared the expression profiles of peripheral blood mononuclear cells (PBMCs) from a single donor with and without pooling with PBMCs isolated from other donors with different blood types. We find that there was no evidence of alloreactivity in the multiplexed samples following three distinct multiplexing workflows (antibody hashing, MULTI-seq, and in silico genotyping using souporcell). Moreover, we identified biases amongst antibody hashing sample classification results in this particular experimental system, as well as gene expression signatures linked to PBMC preparation method (e.g., Ficoll-Paque density gradient centrifugation with or without apheresis using Trima filtration).

scholarly journals Scalable full-transcript coverage single cell RNA sequencing with Smart-seq3xpress

2021 ◽  
Author(s):  
Michael Hagemann-Jensen ◽  
Christoph Ziegenhain ◽  
Rickard Sandberg
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Peripheral Blood Mononuclear Cells ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Cell Types ◽  
Peripheral Blood Mononuclear ◽  
Single Cell Rna Sequencing ◽  
Blood Mononuclear Cells ◽  
Additional Protein

Plate-based single-cell RNA-sequencing methods with full-transcript coverage typically excel at sensitivity but are more resource and time-consuming. Here, we miniaturized and streamlined the Smart-seq3 protocol for drastically reduced cost and increased throughput. Applying Smart-seq3xpress to 16,349 human peripheral blood mononuclear cells revealed a highly granular atlas complete with both common and rare cell types whose identification previously relied on additional protein measurements or the integration with a reference atlas.

scholarly journals The Characteristics of Peripheral Blood Mononuclear Cells in Takayasu Arteritis by Single Cell RNA Sequencing

2021 ◽  
Author(s):  
Qing Gao ◽  
Jinge Yu ◽  
Zuoguan Chen ◽  
Yongpeng Diao ◽  
Yuqing Miao ◽  
...  
Keyword(s):  
B Cells ◽  
Nk Cells ◽  
Single Cell ◽  
Peripheral Blood Mononuclear Cells ◽  
Takayasu Arteritis ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Single Cell Rna Sequencing ◽  
Blood Mononuclear Cells

Objectives Takayasu Arteritis (TA) is a rare non-specific vascular inflammation and has deleterious effects on patients’ health. Recent studies have advanced in TA diagnosis and treatment, but the research on the immune cell atlas of peripheral blood is still less. For this purpose, we performed single-cell RNA sequencing (scRNA-seq) to analyze the inflammatory cell types and cell markers in TA patients’ Peripheral blood mononuclear cells (PBMCs). Methods 4 TA patients and 4 health controls were enrolled in our study from 2019.10 to 2020.5. Their PBMCs samples were collected and performed scRNA-seq. We used Seurat package (v.3.2.2) in R studio (v.3.5.3) for data analysis, and 2 tests were applied for comparing the composition ratio of each cell type by SPSS 20.0. Results CD14+ monocytes, GZMB+ NKT cells, CD56dim CD16+ NK cells, and naive B cells were significantly increased in TA patients as compared to healthy controls and the expression of THBS1, CD163, AREG, IFITM1, TXNIP, and IGHGs was elevated in the peripheral blood of TA patients. Conclusion Except CD4+ T cells, monocytes, NK cells, NKT cells, B cells also play an important role in TA pathogenesis. The elevated markers have different functions in different types of PBMCs, and they can be used as potential diagnostic markers for TA diagnosis.

scholarly journals RNA Sequencing for Gene Expression Profiles in Peripheral Blood Mononuclear Cells with Ankylosing Spondylitis RNA

2020 ◽  
Vol 2020 ◽  
pp. 1-13
Author(s):  
Dan Huang ◽  
Jian Liu ◽  
Yunxiang Cao ◽  
Lei Wan ◽  
Hui Jiang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ankylosing Spondylitis ◽  
Rna Sequencing ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Healthy Controls ◽  
Positive Regulation ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells

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-κB 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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