Single-cell sequencing of the small-RNA transcriptome

Faridani, Omid R.; Abdullayev, Ilgar; Hagemann-Jensen, Michael; Schell, John P.; Lanner, Fredrik; Sandberg, Rickard

doi:10.1038/nbt.3701

Cited by 179 publications

(194 citation statements)

References 153 publications

(157 reference statements)

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“…Efficient first strand synthesis requires a minimal amount of RNA, although techniques exist that reportedly require only miniscule RNA amounts (20). The efficiency of first strand synthesis is not specifically tested in our protocol and has generally not been problematic, because the amount of RNA in a sample can be readily increased by performing multiple reactions.…”

Section: Cdna Library Preparation and Illumina Sequencingmentioning

confidence: 99%

Mapping specificity landscapes of RNA-protein interactions by high throughput sequencing

Jankowsky

Harris

2017

Methods

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To function in a biological setting, RNA binding proteins (RBPs) have to discriminate between alternative binding sites in RNAs. This discrimination can occur in the ground state of an RNA-protein binding reaction, in its transition state, or in both. The extent by which RBPs discriminate at these reaction states defines RBP specificity landscapes. Here, we describe the HiTS-Kin and HiTS-EQ techniques, which combine kinetic and equilibrium binding experiments with high throughput sequencing to quantitatively assess substrate discrimination for large numbers of substrate variants at ground and transition states of RNA-protein binding reactions. We discuss experimental design, practical considerations and data analysis and outline how a combination of HiTS-Kin and HiTS-EQ allows the mapping of RBP specificity landscapes.

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Section: Cdna Library Preparation and Illumina Sequencingmentioning

confidence: 99%

Mapping specificity landscapes of RNA-protein interactions by high throughput sequencing

Jankowsky

Harris

2017

Methods

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“…4d) and to generate the genuine expression profiles of each cell type. Compared with the ligation-based, single-cell sequencing of sRNA developed recently [9], STA has the advantages of a simple procedure, immunity to the 5’-phosphorylation status, and the capability of detecting both noncoding and polyadenylated RNA concurrently. The concurrent detection allows for correlating surface markers with the expression of ncRNAs.…”

Section: Discussionmentioning

confidence: 99%

“…Although it is possible to profile mRNA [8] or miRNA [9] from individual cells, current options to profile both types of RNA concurrently by high-throughput sequencing are limited by the different ways of attaching adapters for reverse transcription (RT) and complementary DNA (cDNA) amplification. The 5’ capping of mRNA is incompatible with the attachment of the 5’ adapter by ligation [10], a procedure commonly used for miRNA amplification.…”

Section: Introductionmentioning

confidence: 99%

Low-cell-number, single-tube amplification (STA) of total RNA revealed transcriptome changes from pluripotency to endothelium

et al. 2017

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BackgroundIn addition to messenger RNA (mRNA), noncoding RNAs (ncRNAs) are essential components in cellular machineries for translation and splicing. Besides their housekeeping functions, ncRNAs are involved in cell type-specific regulation of translation, mRNA stability, genome structure, and accessibility. To have a comprehensive understanding of the identities and functions of different cell types, a method to comprehensively quantify both mRNA and ncRNA in a sensitive manner is highly desirable.MethodsHere we tried to develop a system capable of concurrently profiling both mRNA and ncRNA by polyadenylating RNA in samples before reverse transcription. The sensitivity of the system was maximized by avoiding purification from cell lysis to amplified cDNA and by optimizing the buffer conditions. The single-tube amplification (STA) system was applied to single to 100 cells of 293T cells, human pluripotent stem cells (hPSCs) and their differentiated endothelial progenies to validate its quantitative power and sensitivity by qPCR and high-throughput sequencing.ResultsUsing microRNA (miRNA) as an example, we showed that complementary DNA (cDNA) from ncRNAs could be amplified and specifically detected from a few cells within a single tube. The sensitivity of the system was maximized by avoiding purification from cell lysis to amplified cDNA and by optimizing the buffer conditions. With 100 human embryonic stem cells (hESCs) and their differentiated endothelial cells as input for high-throughput sequencing, the single-tube amplification (STA) system revealed both well-known and other miRNAs selectively enriched in each cell type. The selective enrichment of the miRNAs was further verified by qPCR with 293FT cells and a human induced pluripotent stem cell (hiPSC) line. In addition, the detection of other non-miRNA transcripts indicated that the STA target was not limited to miRNA, but extended to other ncRNAs and mRNAs as well. Finally, the STA system was capable of detecting miRNA and mRNA expression down to single cells, albeit with some loss of sensitivity and power.ConclusionsOverall, STA offered a simple and sensitive way to concurrently quantify both mRNA and ncRNA expression in low-cell-number samples for both qPCR and high-throughput sequencing.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-017-0359-5) contains supplementary material, which is available to authorized users.

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“…Another method, called random displacement amplification sequencing (RamDAseq), has been also developed for non-polyadenylated RNA, including enhancer RNA sequencing [62]. Moreover, Faridani et al [63] designed a method that is specific for small RNA, including miRNA, tRNA, and small nucleolar RNA.…”

Section: Noncoding Rna Sequencing and Profiling In Single Cellsmentioning

confidence: 99%

“…(7) Since scRNA-seq requires a relatively large number of cells for the automatic system of cell separation and capture, it will be challenging when human biopsies from patients are to be used for scRNA-seq. Nevertheless, attempts have been made to solve these problems, e.g., some studies have performed multiple omics analysis in single cells [56][57][58][59][60], and several studies have used specially designed methods to allow non-polyadenylated RNA species to be detected in scRNA-seq [61][62][63].…”

Section: Perspectivesmentioning

confidence: 99%

Understanding the Biology and Pathogenesis of the Kidney by Single-Cell Transcriptomic Analysis

Song

Zhang

et al. 2018

Kidney Dis

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Background: Single-cell RNA-seq (scRNA-seq) has recently emerged as a revolutionary and powerful tool for biomedical research. However, there have been relatively few studies using scRNA-seq in the field of kidney study. Summary: scRNA-seq achieves gene expression profiling at single-cell resolution in contrast with the conventional methods of gene expression profiling, which are based on cell population and give averaged values of gene expression of the cells. Single-cell transcriptomic analysis is crucial because individual cells of the same type are highly heterogeneous in gene expression, which reflects the existence of subpopulations, different cellular states, or molecular dynamics, of the cells, and should be resolved for further insights. In addition, gene expression analysis of tissues or organs that usually comprise multiple cell types or subtypes results in data that are not fully applicable to any given cell type. scRNA-seq is capable of identifying all cell types and subtypes in a tissue, including those that are new or present in small quantity. With these unique capabilities, scRNA-seq has been used to dissect molecular processes in cell differentiation and to trace cell lineages in development. It is also used to analyze the cells in a lesion of disease to identify the cell types and molecular dynamics implicated in the injury. With continuous technical improvement, scRNA-seq has become extremely high throughput and cost effective, making it accessible to all laboratories. In the present review article, we provide an overall review of scRNA-seq concerning its history, improvements, and applications. In addition, we describe the available studies in which scRNA-seq was employed in the field of kidney research. Lastly, we discuss other potential uses of scRNA-seq for kidney research. Key Message: This review article provides general information on scRNA-seq and its various uses. Particularly, we summarize the studies in the field of kidney diseases in which scRNA-seq was used and discuss potential additional uses of scRNA-seq for kidney research.

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Single-cell sequencing of the small-RNA transcriptome

Cited by 179 publications

References 153 publications

Mapping specificity landscapes of RNA-protein interactions by high throughput sequencing

Mapping specificity landscapes of RNA-protein interactions by high throughput sequencing

Low-cell-number, single-tube amplification (STA) of total RNA revealed transcriptome changes from pluripotency to endothelium

Understanding the Biology and Pathogenesis of the Kidney by Single-Cell Transcriptomic Analysis

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