scholarly journals High-throughput RNA sequencing of paraformaldehyde-fixed single cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Hoang Van Phan ◽  
Michiel van Gent ◽  
Nir Drayman ◽  
Anindita Basu ◽  
Michaela U. Gack ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
High Throughput ◽  
Single Cells ◽  
Human Tumor ◽  
Viral Reactivation ◽  
Rna Integrity ◽  
High Throughput Method ◽  
Tumor Virus ◽  
Cell Subpopulations

AbstractSingle-cell transcriptomic studies that require intracellular protein staining, rare cell sorting, or inactivation of infectious pathogens are severely limited. This is because current high-throughput single-cell RNA sequencing methods are either incompatible with or necessitate laborious sample preprocessing for paraformaldehyde treatment, a common tissue and cell fixation and preservation technique. Here we present FD-seq (Fixed Droplet RNA sequencing), a high-throughput method for droplet-based RNA sequencing of paraformaldehyde-fixed, permeabilized and sorted single cells. We show that FD-seq preserves the RNA integrity and relative gene expression levels after fixation and permeabilization. Furthermore, FD-seq can detect a higher number of genes and transcripts than methanol fixation. We first apply FD-seq to analyze a rare subpopulation of cells supporting lytic reactivation of the human tumor virus KSHV, and identify TMEM119 as a potential host factor that mediates viral reactivation. Second, we find that infection with the human betacoronavirus OC43 leads to upregulation of pro-inflammatory pathways in cells that are exposed to the virus but fail to express high levels of viral genes. FD-seq thus enables integrating phenotypic with transcriptomic information in rare cell subpopulations, and preserving and inactivating pathogenic samples.

scholarly journals Fixed single-cell RNA sequencing for understanding virus infection and host response

2020 ◽  
Author(s):  
Hoang Van Phan ◽  
Michiel van Gent ◽  
Nir Drayman ◽  
Anindita Basu ◽  
Michaela U. Gack ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
High Throughput ◽  
Single Cells ◽  
Human Tumor ◽  
Tissue Cell ◽  
Pathogen Inactivation ◽  
Single Cell Rna Sequencing ◽  
Sequencing Studies ◽  
High Level

ABSTRACTSingle-cell RNA sequencing studies requiring intracellular protein staining, rare-cell sorting, or pathogen inactivation are severely limited because current high-throughput methods are incompatible with paraformaldehyde treatment, a very common and simple tissue/cell fixation and preservation technique. Here we present FD-seq, a high-throughput method for droplet-based RNA sequencing of paraformaldehyde-fixed, stained and sorted single-cells. We used FD-seq to address two important questions in virology. First, by analyzing a rare population of cells supporting lytic reactivation of the human tumor virus KSHV, we identified TMEM119 as a host factor that mediates reactivation. Second, we studied the transcriptome of lung cells infected with the coronavirus OC43, which causes the common cold and also serves as a safer model pathogen for SARS-CoV-2. We found that pro-inflammatory pathways are primarily upregulated in abortively-infected or uninfected bystander cells, which are exposed to the virus but fail to express high level of viral genes. FD-seq is suitable for characterizing rare cell populations of interest, for studying high-containment biological samples after inactivation, and for integrating intracellular phenotypic with transcriptomic information.

scholarly journals Cryopreservation of human cancers conserves tumour heterogeneity for single-cell multi-omics analysis

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Sunny Z. Wu ◽  
Daniel L. Roden ◽  
Ghamdan Al-Eryani ◽  
Nenad Bartonicek ◽  
Kate Harvey ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
High Throughput ◽  
Single Cells ◽  
Cellular Heterogeneity ◽  
Tumour Heterogeneity ◽  
Fresh Tissue ◽  
Human Cancers ◽  
Cryopreserved Cell ◽  
Single Cell Rna Sequencing

Abstract Background High throughput single-cell RNA sequencing (scRNA-Seq) has emerged as a powerful tool for exploring cellular heterogeneity among complex human cancers. scRNA-Seq studies using fresh human surgical tissue are logistically difficult, preclude histopathological triage of samples, and limit the ability to perform batch processing. This hindrance can often introduce technical biases when integrating patient datasets and increase experimental costs. Although tissue preservation methods have been previously explored to address such issues, it is yet to be examined on complex human tissues, such as solid cancers and on high throughput scRNA-Seq platforms. Methods Using the Chromium 10X platform, we sequenced a total of ~ 120,000 cells from fresh and cryopreserved replicates across three primary breast cancers, two primary prostate cancers and a cutaneous melanoma. We performed detailed analyses between cells from each condition to assess the effects of cryopreservation on cellular heterogeneity, cell quality, clustering and the identification of gene ontologies. In addition, we performed single-cell immunophenotyping using CITE-Seq on a single breast cancer sample cryopreserved as solid tissue fragments. Results Tumour heterogeneity identified from fresh tissues was largely conserved in cryopreserved replicates. We show that sequencing of single cells prepared from cryopreserved tissue fragments or from cryopreserved cell suspensions is comparable to sequenced cells prepared from fresh tissue, with cryopreserved cell suspensions displaying higher correlations with fresh tissue in gene expression. We showed that cryopreservation had minimal impacts on the results of downstream analyses such as biological pathway enrichment. For some tumours, cryopreservation modestly increased cell stress signatures compared to freshly analysed tissue. Further, we demonstrate the advantage of cryopreserving whole-cells for detecting cell-surface proteins using CITE-Seq, which is impossible using other preservation methods such as single nuclei-sequencing. Conclusions We show that the viable cryopreservation of human cancers provides high-quality single-cells for multi-omics analysis. Our study guides new experimental designs for tissue biobanking for future clinical single-cell RNA sequencing studies.

High-Throughput Production of Single-Cell Microparticles Using an Inkjet Printing Technology

2008 ◽  
Vol 130 (2) ◽  
Author(s):  
Tao Xu ◽  
Helen Kincaid ◽  
Anthony Atala ◽  
James J. Yoo
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Single Cell Analysis ◽  
Single Cells ◽  
Stem Cell Differentiation ◽  
Cell Number ◽  
High Throughput Drug Screening ◽  
Alginate Concentration ◽  
High Throughput Method ◽  
Normal Cellular Function

In this study, a novel biocompatible and inexpensive method for the rapid production of single-cell based microparticles is described. Using an HP DeskJet 550C printer, alginate microparticles containing one to several insulin-producing cells (beta-TC6) were fabricated by coprinting the cells and sodium alginate suspension into a CaCl2 solution. This method is able to generate microparticles of 30–60μm in diameter at a rate as high as 55,000particles∕s. Cell survival assays showed that more than 89% of printed cells survived the fabrication process. The printed beta-TC6 cells demonstrated continuous insulin secretion over a 6day period, which suggests that the printed cells are able to maintain normal cellular function within the microparticles. We show that the printing conditions, such as cell number, alginate concentration, and ionic strengths of CaCl2, influence cellular distribution and geometry of the printed particles. This study presents a simple and high-throughput method to encapsulate single cells, and this technique may be applied in various research investigations, including single-cell analysis, high-throughput drug screening, and stem cell differentiation at the single-cell level.

scholarly journals Missing Data and Technical Variability in Single-Cell RNA- Sequencing Experiments

2015 ◽  
Author(s):  
Stephanie C Hicks ◽  
F. William Townes ◽  
Mingxiang Teng ◽  
Rafael A Irizarry
Keyword(s):  
Gene Expression ◽  
Missing Data ◽  
Single Cell ◽  
Rna Sequencing ◽  
High Throughput ◽  
Single Cells ◽  
Systematic Errors ◽  
Gene Expression Measurement ◽  
Rna Seq ◽  
Batch Effects

Until recently, high-throughput gene expression technology, such as RNA-Sequencing (RNA-seq) required hundreds of thousands of cells to produce reliable measurements. Recent technical advances permit genome-wide gene expression measurement at the single-cell level. Single-cell RNA-Seq (scRNA-seq) is the most widely used and numerous publications are based on data produced with this technology. However, RNA-Seq and scRNA-seq data are markedly different. In particular, unlike RNA-Seq, the majority of reported expression levels in scRNA-seq are zeros, which could be either biologically-driven, genes not expressing RNA at the time of measurement, or technically-driven, gene expressing RNA, but not at a sufficient level to detected by sequencing technology. Another difference is that the proportion of genes reporting the expression level to be zero varies substantially across single cells compared to RNA-seq samples. However, it remains unclear to what extent this cell-to-cell variation is being driven by technical rather than biological variation. Furthermore, while systematic errors, including batch effects, have been widely reported as a major challenge in high-throughput technologies, these issues have received minimal attention in published studies based on scRNA-seq technology. Here, we use an assessment experiment to examine data from published studies and demonstrate that systematic errors can explain a substantial percentage of observed cell-to-cell expression variability. Specifically, we present evidence that some of these reported zeros are driven by technical variation by demonstrating that scRNA-seq produces more zeros than expected and that this bias is greater for lower expressed genes. In addition, this missing data problem is exacerbated by the fact that this technical variation varies cell-to-cell. Then, we show how this technical cell-to-cell variability can be confused with novel biological results. Finally, we demonstrate and discuss how batch-effects and confounded experiments can intensify the problem.

scholarly journals Rapid microfluidic isolation of virally infected primary bronchial epithelial cells for single-cell RNA sequencing

BioTechniques ◽  
2021 ◽  
Vol 71 (1) ◽  
pp. 387-391
Author(s):  
Lucy M Kimbley ◽  
Rachel Parker ◽  
Maaike Sybil Jongen ◽  
John W Holloway ◽  
Emily J Swindle ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Single Cells ◽  
Bronchial Epithelial Cells ◽  
Cdna Libraries ◽  
Bronchial Epithelial ◽  
Rna Integrity ◽  
Single Cell Rna Sequencing ◽  
Primary Bronchial Epithelial Cells

Single-cell RNA sequencing (scRNA-seq) of the bronchial epithelium enables examination of cellular subtypes and their responses to viral infections. Here, an optimized method for the isolation of virally infected primary bronchial epithelial cells using a commercially available microfluidic device is presented. Using this method single cells can be rapidly isolated with minimal equipment available in most laboratories. Isolation can be carried out inside biological safety cabinets, permitting the use of virally infected cells. Both cell-line and primary cells isolated using the device retained sufficient RNA integrity for the generation of short-read sequencing-compatible cDNA libraries to facilitate scRNA-seq.

scholarly journals Ultra-high-throughput single-cell RNA sequencing and perturbation screening with combinatorial fluidic indexing

2021 ◽  
Author(s):  
Paul Datlinger ◽  
André F. Rendeiro ◽  
Thorina Boenke ◽  
Martin Senekowitsch ◽  
Thomas Krausgruber ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
High Throughput ◽  
Single Cell Rna Sequencing

scholarly journals P02.10 FocuSCOPE: a single cell, multi-omics solution to simultaneously analyze tumor variants and microenvironment

2021 ◽  
Vol 9 (Suppl 1) ◽  
pp. A12.1-A12
Author(s):  
Y Arjmand Abbassi ◽  
N Fang ◽  
W Zhu ◽  
Y Zhou ◽  
Y Chen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Tumor Microenvironment ◽  
Single Cell ◽  
High Throughput ◽  
Immune Cells ◽  
Genetic Variants ◽  
Expression Profiles ◽  
Single Cells ◽  
Gene Expression Profiles ◽  
Single Cell Sequencing

Recent advances of high-throughput single cell sequencing technologies have greatly improved our understanding of the complex biological systems. Heterogeneous samples such as tumor tissues commonly harbor cancer cell-specific genetic variants and gene expression profiles, both of which have been shown to be related to the mechanisms of disease development, progression, and responses to treatment. Furthermore, stromal and immune cells within tumor microenvironment interact with cancer cells to play important roles in tumor responses to systematic therapy such as immunotherapy or cell therapy. However, most current high-throughput single cell sequencing methods detect only gene expression levels or epigenetics events such as chromatin conformation. The information on important genetic variants including mutation or fusion is not captured. To better understand the mechanisms of tumor responses to systematic therapy, it is essential to decipher the connection between genotype and gene expression patterns of both tumor cells and cells in the tumor microenvironment. We developed FocuSCOPE, a high-throughput multi-omics sequencing solution that can detect both genetic variants and transcriptome from same single cells. FocuSCOPE has been used to successfully perform single cell analysis of both gene expression profiles and point mutations, fusion genes, or intracellular viral sequences from thousands of cells simultaneously, delivering comprehensive insights of tumor and immune cells in tumor microenvironment at single cell resolution.Disclosure InformationY. Arjmand Abbassi: None. N. Fang: None. W. Zhu: None. Y. Zhou: None. Y. Chen: None. U. Deutsch: None.

scholarly journals 192 Pooled T cell receptor screening (POTS) provides unbiased, high-throughput method for TCR discovery

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A204-A204
Author(s):  
Jack Reid ◽  
Shihong Zhang ◽  
Ariunaa Munkhbat ◽  
Matyas Ecsedi ◽  
Megan McAfee ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
T Cell Receptor ◽  
High Throughput ◽  
Ex Vivo ◽  
Cell Receptor ◽  
High Avidity ◽  
Reporter System ◽  
High Throughput Method

BackgroundT Cell Receptor (TCR)-T cell therapies have shown some promising results in cancer clinical trials, however the efficacy of treatment remains suboptimal. Outcomes could potentially be improved by utilizing highly functional TCRs for future trials. Current TCR discovery methods are relatively low throughput and rely on synthesis and screening of individual TCRs based on tetramer binding and peptide specificity, which is costly and labor intensive. We have developed and validated a pooled approach relying on directly cloned TCRs transduced into a fluorescent Jurkat reporter system (figure 1). This approach provides an unbiased, high-throughput method for TCR discovery.MethodsAs a model for POTS, T cells specific for a peptide derived adenovirus structural protein were sorted on tetramer and subjected to 10x single cell VDJ analysis. Pools of randomly paired TCR alpha and beta chains were cloned from the 10x cDNA into a lentiviral vector and transduced into a Jurkat reporter cells. Consecutive stimulations with cognate antigen followed by cell sorts were performed to enrich for functional TCRs. Full length TCRab pools were sequenced by Oxford Nanopore Technologies (ONT) and compared to a 10x dataset to find naturally paired TCRs.ResultsComparison between the ex vivo single cell VDJ sequencing and ONT sequencing of the transduced antigen specific TCRs showed more than 99% of the TCR pairs found in reporter positive Jurkat cells were naturally paired TCRs. The functionality of 8 TCR clonotypes discovered using POTS were compared and clone #2 showed the strongest response. Of the selected clonotypes, clone #2 showed a low frequency of 0.9% in the ex vivo single cell VDJ sequencing. After the first round of stimulation and sequencing, clone #2 takes up of 5% of all reporter-positive clones. The abundance of clone #2 further increased to 17% after another round of stimulation, sorting and sequencing, suggesting this method can retrieve and enrich for highly functional antigen specific TCRs.Abstract 192 Figure 1Outline of the POTS workflow.ConclusionsPOTS provides a high-throughput method for discovery of naturally paired, high-avidity T cell receptors. This method mitigates bias introduced by T cell differentiation state by screening TCRs in a clonal reporter system. Additionally, POTS allows for screening of low abundance clones when compared with traditional TCR discovery techniques. Pooled TCRs could also be screened in vivo with primary T cells in a mouse model to screen for the most functional and physiologically fit TCR for cancer treatment.

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