Multiplexed single-cell RNA-seq via transient barcoding for drug screening

AbstractTo simultaneously analyze multiple samples of various conditions with scRNA-seq, we developed a universal sample barcoding method through transient transfection of SBOs. A 48-plex drug treatment experiment of pooled samples analyzed by a single run of Drop-Seq revealed a unique transcriptome response for each drug and target-specific gene expression signatures at the single-cell level. Our cost-effective method is widely applicable for single-cell profiling of multiple experimental conditions.

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Multiplexed single-cell RNA-seq via transient barcoding for simultaneous expression profiling of various drug perturbations

Science Advances ◽

10.1126/sciadv.aav2249 ◽

2019 ◽

Vol 5 (5) ◽

pp. eaav2249 ◽

Cited By ~ 22

Author(s):

Dongju Shin ◽

Wookjae Lee ◽

Ji Hyun Lee ◽

Duhee Bang

Keyword(s):

Gene Expression ◽

Single Cell ◽

Cost Effective ◽

Specific Gene ◽

Rna Seq ◽

Experimental Conditions ◽

Cost Effective Method ◽

Treatment Experiment ◽

Single Cell Profiling ◽

Multiple Samples

The development of high-throughput single-cell RNA sequencing (scRNA-seq) has enabled access to information about gene expression in individual cells and insights into new biological areas. Although the interest in scRNA-seq has rapidly grown in recent years, the existing methods are plagued by many challenges when performing scRNA-seq on multiple samples. To simultaneously analyze multiple samples with scRNA-seq, we developed a universal sample barcoding method through transient transfection with short barcode oligonucleotides. By conducting a species-mixing experiment, we have validated the accuracy of our method and confirmed the ability to identify multiplets and negatives. Samples from a 48-plex drug treatment experiment were pooled and analyzed by a single run of Drop-Seq. This revealed unique transcriptome responses for each drug and target-specific gene expression signatures at the single-cell level. Our cost-effective method is widely applicable for the single-cell profiling of multiple experimental conditions, enabling the widespread adoption of scRNA-seq for various applications.

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Vireo: Bayesian demultiplexing of pooled single-cell RNA-seq data without genotype reference

Genome Biology ◽

10.1186/s13059-019-1865-2 ◽

2019 ◽

Vol 20 (1) ◽

Cited By ~ 13

Author(s):

Yuanhua Huang ◽

Davis J. McCarthy ◽

Oliver Stegle

Keyword(s):

Single Cell ◽

Real Data ◽

Experimental Designs ◽

Rna Seq ◽

Computationally Efficient ◽

Primary Object ◽

Genotype Information ◽

Object Of Study ◽

Multiple Samples ◽

Pooled Samples

AbstractMultiplexed single-cell RNA-seq analysis of multiple samples using pooling is a promising experimental design, offering increased throughput while allowing to overcome batch variation. To reconstruct the sample identify of each cell, genetic variants that segregate between the samples in the pool have been proposed as natural barcode for cell demultiplexing. Existing demultiplexing strategies rely on availability of complete genotype data from the pooled samples, which limits the applicability of such methods, in particular when genetic variation is not the primary object of study. To address this, we here present Vireo, a computationally efficient Bayesian model to demultiplex single-cell data from pooled experimental designs. Uniquely, our model can be applied in settings when only partial or no genotype information is available. Using pools based on synthetic mixtures and results on real data, we demonstrate the robustness of Vireo and illustrate the utility of multiplexed experimental designs for common expression analyses.

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A statistical framework for differential pseudotime analysis with multiple single-cell RNA-seq samples

10.1101/2021.07.10.451910 ◽

2021 ◽

Author(s):

Wenpin Hou ◽

Zhicheng Ji ◽

Zeyu Chen ◽

E John Wherry ◽

Stephanie C Hicks ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Biological Processes ◽

Rna Seq ◽

Experimental Conditions ◽

Computational Framework ◽

Statistical Framework ◽

Gene Regulatory ◽

Multiple Samples ◽

False Discoveries

Pseudotime analysis with single-cell RNA-sequencing (scRNA-seq) data has been widely used to study dynamic gene regulatory programs along continuous biological processes. While many computational methods have been developed to infer the pseudo-temporal trajectories of cells within a biological sample, methods that compare pseudo-temporal patterns with multiple samples (or replicates) across different experimental conditions are lacking. Lamian is a comprehensive and statistically-rigorous computational framework for differential multi-sample pseudotime analysis. It can be used to identify changes in a biological process associated with sample covariates, such as different biological conditions, and also to detect changes in gene expression, cell density, and topology of a pseudotemporal trajectory. Unlike existing methods that ignore sample variability, Lamian draws statistical inference after accounting for cross-sample variability and hence substantially reduces sample-specific false discoveries that are not generalizable to new samples. Using both simulations and real scRNA-seq data, including an analysis of differential immune response programs between COVID-19 patients with different disease severity levels, we demonstrate the advantages of Lamian in decoding cellular gene expression programs in continuous biological processes.

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Facile Fabrication of Biochar from Palm Kernel Shell Waste and Its Novel Application to Magnesium-Based Materials for Hydrogen Storage

Materials ◽

10.3390/ma13030625 ◽

2020 ◽

Vol 13 (3) ◽

pp. 625 ◽

Cited By ~ 2

Author(s):

Martin Luther Yeboah ◽

Xinyuan Li ◽

Shixue Zhou

Keyword(s):

Hydrogen Storage ◽

Palm Kernel ◽

Cost Effective ◽

Ambient Air ◽

Decomposition Temperature ◽

Scanning Calorimetry ◽

Experimental Conditions ◽

Palm Kernel Shell ◽

Cost Effective Method ◽

Facile Fabrication

In this investigation, an easily-operated and cost-effective method is utilized to synthesize biochar in ambient air, and the prepared biochar is used in a novel manner as a milling aid for fabricating Mg-biochar composites for hydrogen storage. X-ray diffractometry reveals that increasing the content of palm kernel shell biochar (PKSBC) from 5 wt.% to 20 wt.% enhances the hydrogen absorption performance by increasing the conversion of Mg into MgH2 from 83% to 93%. A 40 °C reduction in decomposition temperature of MgH2 is recorded from differential scanning calorimetry curves when the content of PKSBC is increased to 20 wt.%. Magnesium is milled and hydrided under the same experimental conditions and used as a reference material. It is proposed that these property enhancements can be attributed to the fact that PKSBC acts as an anti-sticking agent for elemental Mg powders, helping in the achievement of a more dispersed composite with reduced Mg particle size due to its layered-like carbon structure.

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MultiEditR: An easy validation method for detecting and quantifying RNA editing from Sanger sequencing

10.1101/633685 ◽

2019 ◽

Cited By ~ 4

Author(s):

Mitchell Kluesner ◽

Annette Arnold ◽

Taga Lerner ◽

Rafail Nikolaos Tasakis ◽

Sandra Wüst ◽

...

Keyword(s):

Rna Editing ◽

Sanger Sequencing ◽

Tumour Progression ◽

Cost Effective ◽

Base Change ◽

Rna Seq ◽

Multiple Sources ◽

Base Editing ◽

Cost Effective Method ◽

Apobec Family

ABSTRACTRNA editing is the base change that results from RNA deamination by two predominant classes of deaminases; the APOBEC family and the ADAR family. Respectively, deamination of nucleobases by these enzymes are responsible for endogenous editing of cytosine to uracil (C-to-U) and adenosine to inosine (A-to-I). RNA editing is known to play an essential role both in maintaining normal cellular function, as well as altered cellular physiology during oncogenesis and tumour progression. Analysis of RNA editing in these important processes, largely relies on RNA-seq technology for the detection and quantification of RNA editing sites. Despite the power of these technologies, multiple sources of error in detecting and measuring base editing still exist, therefore additional validation and quantification of editing through Sanger sequencing is still required for confirmation of editing. Depending on the number of RNA editing sites that are of interest, this validation step can be both expensive and time-consuming. To address this need we developed the tool MultiEditR which provides a simple, and cost-effective method of detecting and quantifying RNA editing form Sanger sequencing. We expect that MultiEditR will foster further discoveries in this rapidly expanding field.

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Ultra-high throughput single-cell RNA sequencing by combinatorial fluidic indexing

10.1101/2019.12.17.879304 ◽

2019 ◽

Cited By ~ 4

Author(s):

Paul Datlinger ◽

André F Rendeiro ◽

Thorina Boenke ◽

Thomas Krausgruber ◽

Daniele Barreca ◽

...

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Population Genomics ◽

Cost Effective ◽

Mouse Cell ◽

Droplet Microfluidics ◽

Rna Seq ◽

Single Cell Rna Sequencing ◽

Massive Scale ◽

Tcr Activation

AbstractCell atlas projects and single-cell CRISPR screens hit the limits of current technology, as they require cost-effective profiling for millions of individual cells. To satisfy these enormous throughput requirements, we developed “single-cell combinatorial fluidic indexing” (scifi) and applied it to single-cell RNA sequencing. The resulting scifi-RNA-seq assay combines one-step combinatorial pre-indexing of single-cell transcriptomes with subsequent single-cell RNA-seq using widely available droplet microfluidics. Pre-indexing allows us to load multiple cells per droplet, which increases the throughput of droplet-based single-cell RNA-seq up to 15-fold, and it provides a straightforward way of multiplexing hundreds of samples in a single scifi-RNA-seq experiment. Compared to multi-round combinatorial indexing, scifi-RNA-seq provides an easier, faster, and more efficient workflow, thereby enabling massive-scale scRNA-seq experiments for a broad range of applications ranging from population genomics to drug screens with scRNA-seq readout. We benchmarked scifi-RNA-seq on various human and mouse cell lines, and we demonstrated its feasibility for human primary material by profiling TCR activation in T cells.

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Bulk Tissue Cell Type Deconvolution with Multi-Subject Single-Cell Expression Reference

10.1101/354944 ◽

2018 ◽

Cited By ~ 1

Author(s):

Xuran Wang ◽

Jihwan Park ◽

Katalin Susztak ◽

Nancy R. Zhang ◽

Mingyao Li

Keyword(s):

Single Cell ◽

Cell Types ◽

Cellular Heterogeneity ◽

Tissue Cell ◽

Specific Gene ◽

Rna Seq ◽

Cell Type ◽

Cell Type Specific ◽

Cell Expression

AbstractWe present MuSiC, a method that utilizes cell-type specific gene expression from single-cell RNA sequencing (RNA-seq) data to characterize cell type compositions from bulk RNA-seq data in complex tissues. When applied to pancreatic islet and whole kidney expression data in human, mouse, and rats, MuSiC outperformed existing methods, especially for tissues with closely related cell types. MuSiC enables characterization of cellular heterogeneity of complex tissues for identification of disease mechanisms.

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DeepDRIM: a deep neural network to reconstruct cell-type-specific gene regulatory network using single-cell RNA-Seq Data

10.1101/2021.02.03.429484 ◽

2021 ◽

Author(s):

Jiaxing Chen ◽

Chinwang Cheong ◽

Liang Lan ◽

Xin Zhou ◽

Jiming Liu ◽

...

Keyword(s):

Neural Network ◽

Single Cell ◽

Regulatory Networks ◽

Deep Neural Network ◽

Neighborhood Context ◽

Cellular Heterogeneity ◽

Specific Gene ◽

Rna Seq ◽

Cell Type Specific ◽

Gene Regulatory

AbstractSingle-cell RNA sequencing is used to capture cell-specific gene expression, thus allowing reconstruction of gene regulatory networks. The existing algorithms struggle to deal with dropouts and cellular heterogeneity, and commonly require pseudotime-ordered cells. Here, we describe DeepDRIM a supervised deep neural network that represents gene pair joint expression as images and considers the neighborhood context to eliminate the transitive interactions. Deep-DRIM yields significantly better performance than the other nine algorithms used on the eight cell lines tested, and can be used to successfully discriminate key functional modules between patients with mild and severe symptoms of coronavirus disease 2019 (COVID-19).

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Sashimi plots: Quantitative visualization of alternative isoform expression from RNA-seq data

10.1101/002576 ◽

2014 ◽

Cited By ~ 3

Author(s):

Yarden Katz ◽

Eric T Wang ◽

Jacob Stilterra ◽

Schraga Schwartz ◽

Bang Wong ◽

...

Keyword(s):

Region Of Interest ◽

Splice Junction ◽

Genomic Region ◽

Rna Seq ◽

Mrna Isoforms ◽

Experimental Conditions ◽

Human Genes ◽

Quantitative Visualization ◽

Isoform Expression ◽

Multiple Samples

Analysis of RNA sequencing (RNA-Seq) data revealed that the vast majority of human genes express multiple mRNA isoforms, produced by alternative pre-mRNA splicing and other mechanisms, and that most alternative isoforms vary in expression between human tissues. As RNA-Seq datasets grow in size, it remains challenging to visualize isoform expression across multiple samples. We present Sashimi plots, a quantitative multi-sample visualization of RNA-Seq reads aligned to gene annotations, which enables quantitative comparison of isoform usage across samples or experimental conditions. Given an input annotation and spliced alignments of reads from a sample, a region of interest is visualized in a Sashimi plot as follows: (i) alignments in exons are represented as read densities (optionally normalized by length of genomic region and coverage), and (ii) splice junction reads are drawn as arcs connecting a pair of exons, where arc width is drawn proportional to the number of reads aligning to the junction.

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Comprehensive single cell transcriptional profiling of a multicellular organism by combinatorial indexing

10.1101/104844 ◽

2017 ◽

Cited By ~ 8

Author(s):

Junyue Cao ◽

Jonathan S. Packer ◽

Vijay Ramani ◽

Darren A. Cusanovich ◽

Chau Huynh ◽

...

Keyword(s):

Single Cell ◽

Expression Profiles ◽

Single Cells ◽

Transcriptional Profiling ◽

Cost Effective ◽

Cell Types ◽

Cell Capture ◽

Rna Seq ◽

Major Step ◽

C Elegans

AbstractConventional methods for profiling the molecular content of biological samples fail to resolve heterogeneity that is present at the level of single cells. In the past few years, single cell RNA sequencing has emerged as a powerful strategy for overcoming this challenge. However, its adoption has been limited by a paucity of methods that are at once simple to implement and cost effective to scale massively. Here, we describe a combinatorial indexing strategy to profile the transcriptomes of large numbers of single cells or single nuclei without requiring the physical isolation of each cell (Single cell Combinatorial Indexing RNA-seq or sci-RNA-seq). We show that sci-RNA-seq can be used to efficiently profile the transcriptomes of tens-of-thousands of single cells per experiment, and demonstrate that we can stratify cell types from these data. Key advantages of sci-RNA-seq over contemporary alternatives such as droplet-based single cell RNA-seq include sublinear cost scaling, a reliance on widely available reagents and equipment, the ability to concurrently process many samples within a single workflow, compatibility with methanol fixation of cells, cell capture based on DNA content rather than cell size, and the flexibility to profile either cells or nuclei. As a demonstration of sci-RNA-seq, we profile the transcriptomes of 42,035 single cells from C. elegans at the L2 stage, effectively 50-fold “shotgun cellular coverage” of the somatic cell composition of this organism at this stage. We identify 27 distinct cell types, including rare cell types such as the two distal tip cells of the developing gonad, estimate consensus expression profiles and define cell-type specific and selective genes. Given that C. elegans is the only organism with a fully mapped cellular lineage, these data represent a rich resource for future methods aimed at defining cell types and states. They will advance our understanding of developmental biology, and constitute a major step towards a comprehensive, single-cell molecular atlas of a whole animal.

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