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

Picelli, Simone; Faridani, Omid R.; Björklund, Åsa K.; Winberg, Gösta; Sagasser, Sven; Sandberg, Rickard

doi:10.1038/nprot.2014.006

Cited by 3,534 publications

(3,121 citation statements)

References 34 publications

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“…We validated our droplet-based data by independently analyzing 1,522 epithelial cells using full-length scRNA-seq 16 , with much higher coverage per cell (Fig. 1a, Extended Data Fig.…”

Section: Resultsmentioning

confidence: 90%

“…Libraries were prepared using a modified SMART-Seq2 protocol as previously reported 16 . Briefly, RNA lysate cleanup was preformed using RNAClean XP beads (Agencourt) followed by reverse transcription with Maxima Reverse Transcriptase (Life Technologies) and whole transcription amplification (WTA) with KAPA HotStart HIFI 2× ReadyMix (Kapa Biosystems) for 21 cycles.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

A single-cell survey of the small intestinal epithelium

Haber

Biton

Rogel

et al. 2017

Nature

1,416

115

1,694

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Intestinal epithelial cells (IECs) absorb nutrients, respond to microbes, provide barrier function and help coordinate immune responses. We profiled 53,193 individual epithelial cells from mouse small intestine and organoids, and characterized novel subtypes and their gene signatures. We showed unexpected diversity of hormone-secreting enteroendocrine cells and constructed their novel taxonomy. We distinguished between two tuft cell subtypes, one of which expresses the epithelial cytokine TSLP and CD45 (Ptprc), the pan-immune marker not previously associated with non-hematopoietic cells. We also characterized how cell-intrinsic states and cell proportions respond to bacterial and helminth infections. Salmonella infection caused an increase in Paneth cells and enterocytes abundance, and broad activation of an antimicrobial program. In contrast, Heligmosomoides polygyrus caused an expansion of goblet and tuft cell populations. Our survey highlights new markers and programs, associates sensory molecules to cell types, and uncovers principles of gut homeostasis and response to pathogens.

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“…We validated our droplet-based data by independently analyzing 1,522 epithelial cells using full-length scRNA-seq 16 , with much higher coverage per cell (Fig. 1a, Extended Data Fig.…”

Section: Resultsmentioning

confidence: 90%

Section: Methodsmentioning

confidence: 99%

A single-cell survey of the small intestinal epithelium

Haber

Biton

Rogel

et al. 2017

Nature

1,416

115

1,694

View full text Add to dashboard Cite

show abstract

“…RNA Sequencing and Mapping of Reads mRNA from the single cells was amplified using the SMARTSeq2 protocol (Picelli et al, 2014), with the additional inclusion of ERCC spike-in control (1 ml of 1:250,000 [batches 1 and 2] or a 1:1,000,000 [batch 3 and batch 4] dilution of mix 1 [Ambion] per cell). Multiplex sequencing libraries were generated from amplified cDNA using Nextera XT (Illumina) and sequenced on a HiSeq 2500 running in rapid mode.…”

Section: Scoring 2-to 4-cell Division Patternmentioning

confidence: 99%

Heterogeneity in Oct4 and Sox2 Targets Biases Cell Fate in 4-Cell Mouse Embryos

Goolam

Scialdone

Graham

et al. 2016

Obstetrical &Amp; Gynecological Survey

125

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SUMMARYThe major and essential objective of pre-implantation development is to establish embryonic and extra-embryonic cell fates. To address when and how this fundamental process is initiated in mammals, we characterize transcriptomes of all individual cells throughout mouse pre-implantation development. This identifies targets of master pluripotency regulators Oct4 and Sox2 as being highly heterogeneously expressed between blastomeres of the 4-cell embryo, with Sox21 showing one of the most heterogeneous expression profiles. Live-cell tracking demonstrates that cells with decreased Sox21 yield more extra-embryonic than pluripotent progeny. Consistently, decreasing Sox21 results in premature upregulation of the differentiation regulator Cdx2, suggesting that Sox21 helps safeguard pluripotency. Furthermore, Sox21 is elevated following increased expression of the histone H3R26-methylase CARM1 and is lowered following CARM1 inhibition, indicating the importance of epigenetic regulation. Therefore, our results indicate that heterogeneous gene expression, as early as the 4-cell stage, initiates cell-fate decisions by modulating the balance of pluripotency and differentiation.

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“…In the past few years a wealth of techniques have been developed to study genome-wide transcriptional output at the single-cell level [1][2][3][4][5][6][7]. In contrast to methods relying on sequencing or PCR, image-based transcriptomics visualizes single transcripts in a population of single cells in situ.…”

Section: Image-based Transcriptomics Is Unique In Several Waysmentioning

confidence: 99%

Computer vision for image-based transcriptomics

Stoeger

Battich

Herrmann

et al. 2015

Methods

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Single-cell transcriptomics has recently emerged as one of the most promising tools for understanding the diversity of the transcriptome among single cells. Image-based transcriptomics is unique compared to other methods as it does not require conversion of RNA to cDNA prior to signal amplification and transcript quantification. Thus, its efficiency in transcript detection is unmatched by other methods. In addition, image-based transcriptomics allows the study of the spatial organization of the transcriptome in single cells at single-molecule, and, when combined with superresolution microscopy, nanometer resolution. However, in order to unlock the full power of image-based transcriptomics, robust computer vision of single molecules and cells is required. Here, we shortly discuss the setup of the experimental pipeline for image-based transcriptomics, and then describe in detail the algorithms that we developed to extract, at high-throughput, robust multivariate feature sets of transcript molecule abundance, localization and patterning in tens of thousands of single cells across the transcriptome. These computer vision algorithms and pipelines can be downloaded from: https://github.com/pelkmanslab/ImageBasedTranscriptomics. AbstractSingle-cell transcriptomics has recently emerged as one of the most promising tools for

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

Cited by 3,534 publications

References 34 publications

A single-cell survey of the small intestinal epithelium

A single-cell survey of the small intestinal epithelium

Heterogeneity in Oct4 and Sox2 Targets Biases Cell Fate in 4-Cell Mouse Embryos

Computer vision for image-based transcriptomics

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