mRNA-Seq whole-transcriptome analysis of a single cell

  title={mRNA-Seq whole-transcriptome analysis of a single cell},
  author={Fuchou Tang and Catalin C. Barbacioru and Yangzhou Wang and Ellen Nordman and Clarence C Lee and Nanlan Xu and Xiaohui Wang and John P. Bodeau and Brian B. Tuch and Asim S. Siddiqui and Kaiqin Lao and M. Azim Surani},
  journal={Nature Methods},
Next-generation sequencing technology is a powerful tool for transcriptome analysis. However, under certain conditions, only a small amount of material is available, which requires more sensitive techniques that can preferably be used at the single-cell level. Here we describe a single-cell digital gene expression profiling assay. Using our mRNA-Seq assay with only a single mouse blastomere, we detected the expression of 75% (5,270) more genes than microarray techniques and identified 1,753… 

CEL-Seq: single-cell RNA-Seq by multiplexed linear amplification.

Single-cell Analysis Of Mrna Splicing Variants

This work attempts to expand the toolbox of spatially informed RNA visualization methods by optimizing click-amplifying fluorescent in situ hybridization (clampFISH) to visualize alternatively-spliced isoforms and describes some of the challenges it encountered.

RNA-seq based transcriptomic analysis of single bacterial cells.

Preliminary analysis of single-cell transcriptomic datasets revealed that genes from the "Mobile elements" functional category have the most significant increase of gene-expression heterogeneity upon stress, which was further confirmed by single- cell RT-qPCR analysis of gene expression in 24 randomly selected cells.

Full-Length mRNA-Seq from single cell levels of RNA and individual circulating tumor cells

Applying Smart-Seq to circulating tumor cells from melanomas, it is found that although gene expression estimates from single cells have increased noise, hundreds of differentially expressed genes could be identified using few cells per cell type.

RNA-Seq analysis to capture the transcriptome landscape of a single cell

This protocol can generate deep-sequencing libraries for 16 single-cell samples within 6 d and can capture up to 75% more genes expressed in early embryos compared with cDNA microarray techniques.

Full-length RNA-seq from single cells using Smart-seq2

A detailed protocol is presented for Smart-seq2 that allows the generation of full-length cDNA and sequencing libraries by using standard reagents and the lack of strand specificity and the inability to detect nonpolyadenylated (polyA−) RNA.

Droplet-based Single-cell Total RNA-seq Reveals Differential Non-Coding Expression and Splicing Patterns during Mouse Development

VASA-seq is developed to detect the total transcriptome in single cells and provides the first comprehensive analysis of alternative splicing during mammalian development and is compatible with both plate-based formats and droplet microfluidics.

SC3-seq: a method for highly parallel and quantitative measurement of single-cell gene expression

The SC3-seq reveals the heterogeneity in human-induced pluripotent stem cells (hiPSCs) cultured under on-feeder as well as feeder-free conditions, demonstrating a more homogeneous property of the feeder -free hiPSCs.

Evaluation of tools for highly variable gene discovery from single-cell RNA-seq data

The results demonstrate that reproducibility in HVG analysis requires a larger sample size than DEG analysis, and seven HVG methods from six software packages, including BASiCS, Brennecke, scLVM, scran, scVEGs and Seurat are compared.

The review of transcriptome sequencing: principles, history and advances

  • Haotian Zhang
  • Biology
    IOP Conference Series: Earth and Environmental Science
  • 2019
Single cell RNA-seq method has been developed to better characterize the transcriptomes of various cell types in biological tissues and reveal the heterogeneity of gene expression between cells.



Stem cell transcriptome profiling via massive-scale mRNA sequencing

A massive-scale RNA sequencing protocol, short quantitative random RNA libraries or SQRL, is developed, highlighting how SQRL can be used to characterize transcriptome content and dynamics in a quantitative and reproducible manner, and suggesting that the understanding of transcriptional complexity is far from complete.

An improved single-cell cDNA amplification method for efficient high-density oligonucleotide microarray analysis

A strategy to globally amplify mRNAs from single cells for highly quantitative high-density oligonucleotide microarray analysis that combines a small number of directional PCR cycles with subsequent linear amplification successfully detects subtle but essential differences in gene expression at the single-cell level among seemingly homogeneous cell populations is reported.

RNA-seq: an assessment of technical reproducibility and comparison with gene expression arrays.

It is found that the Illumina sequencing data are highly replicable, with relatively little technical variation, and thus, for many purposes, it may suffice to sequence each mRNA sample only once (i.e., using one lane).

Mapping and quantifying mammalian transcriptomes by RNA-Seq

Although >90% of uniquely mapped reads fell within known exons, the remaining data suggest new and revised gene models, including changed or additional promoters, exons and 3′ untranscribed regions, as well as new candidate microRNA precursors.

A Global View of Gene Activity and Alternative Splicing by Deep Sequencing of the Human Transcriptome

A global survey of messenger RNA splicing events identified 94,241 splice junctions and showed that exon skipping is the most prevalent form of alternative splicing.

Analysis of G protein α subunit mRNA abundance in preimplantation mouse embryos using a rapid, quantitative RT‐PCR approach

A novel reverse transcription‐polymerase chain reaction (RT‐PCR)‐based approach for systematically quantifying in a single experiment the abundances of many different mRNAs in preimplantation mouse embryos, which provides reliable, quantitative data regarding changes in mRNA abundance.

Global single-cell cDNA amplification to provide a template for representative high-density oligonucleotide microarray analysis

A protocol for the representative amplification of global mRNAs from typical single mammalian cells to provide a template for high-density oligonucleotide microarray analysis and yields cDNA templates sufficient for more than 80 microarray hybridizations from a single cell.

Determination of tag density required for digital transcriptome analysis: Application to an androgen-sensitive prostate cancer model

A double-random priming method for deep sequencing to profile double poly(A)-selected RNA from LNCaP cells before and after androgen stimulation is described and a general guide for analysis of gene expression and alternative splicing by deep sequencing is provided.

Alternative Isoform Regulation in Human Tissue Transcriptomes

An in-depth analysis of 15 diverse human tissue and cell line transcriptomes on the basis of deep sequencing of complementary DNA fragments yielding a digital inventory of gene and mRNA isoform expression suggested common involvement of specific factors in tissue-level regulation of both splicing and polyadenylation.

RNA-Seq: a revolutionary tool for transcriptomics

The RNA-Seq approach to transcriptome profiling that uses deep-sequencing technologies provides a far more precise measurement of levels of transcripts and their isoforms than other methods.