Laser capture microscopy coupled with Smart-seq2 for precise spatial transcriptomic profiling

Nichterwitz, Susanne; Chen, Geng; Benitez, Julio Aguila; Yilmaz, Marlene; Storvall, Helena; Cao, Mengshu; Sandberg, Rickard; Deng, Qiaolin; Hedlund, Eva

doi:10.1038/ncomms12139

Cited by 276 publications

(310 citation statements)

References 38 publications

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“…Whereas initial studies analyzed a handful to a hundred cells 13,14,17,55 , a series of technological advances, from robotics 16,56 to microfluidics 39,57 to reverse emulsion and hydrogel droplets increased assay throughput to tens or even hundreds of thousands of cells in an experiment 58,59 . There were also advances in techniques for acquiring cells and processing minuscule amounts of RNA 14,60 , improving the robustness of scRNA-seq on small samples, such as biopsies 54,61 , fixed cells 62 , and even avoiding tissue dissociation altogether by isolating nuclei 48,63 . It is still not routine to apply scRNA-seq to clinical samples, but it is certainly within reach 38,54 .…”

Section: Single Cell Genomics: State Of the Artmentioning

confidence: 99%

Scaling single-cell genomics from phenomenology to mechanism

Tanay

Regev

2017

Nature

683

560

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Three fundamental questions in biology are how do individual cells differentiate to form tissues, how do tissues function in a coordinated and flexible fashion, and which gene regulatory mechanisms support these processes. Single cell genomics open new ways to tackle these questions by combining the comprehensive nature of genomics with the microscopic resolution required to describe complex multi-cellular systems. Early single cell genomic studies provides us with remarkably rich phenomenology of heterogeneous cellular states, but transforming observational studies to models of dynamics and causal mechanism in tissues poses new challenges and require stronger integration between theoretical, computational and experimental frameworks.

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Section: Single Cell Genomics: State Of the Artmentioning

confidence: 99%

Scaling single-cell genomics from phenomenology to mechanism

Tanay

Regev

2017

Nature

683

560

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“…The protocol has been previously described in detail (Allodi and Hedlund 2014;Nichterwitz et al 2016). …”

Section: Extended Culture Of Female Delayed Episcsmentioning

confidence: 99%

Single-cell analyses of X Chromosome inactivation dynamics and pluripotency during differentiation

et al. 2016

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Pluripotency, differentiation, and X Chromosome inactivation (XCI) are key aspects of embryonic development. However, the underlying relationship and mechanisms among these processes remain unclear. Here, we systematically dissected these features along developmental progression using mouse embryonic stem cells (mESCs) and single-cell RNA sequencing with allelic resolution. We found that mESCs grown in a ground state 2i condition displayed transcriptomic profiles diffused from preimplantation mouse embryonic cells, whereas EpiStem cells closely resembled the post-implantation epiblast. Sex-related gene expression varied greatly across distinct developmental states. We also identified novel markers that were highly enriched in each developmental state. Moreover, we revealed that several novel pathways, including PluriNetWork and Focal Adhesion, were responsible for the delayed progression of female EpiStem cells. Importantly, we "digitalized" XCI progression using allelic expression of active and inactive X Chromosomes and surprisingly found that XCI states exhibited profound variability in each developmental state, including the 2i condition. XCI progression was not tightly synchronized with loss of pluripotency and increase of differentiation at the single-cell level, although these processes were globally correlated. In addition, highly expressed genes, including core pluripotency factors, were in general biallelically expressed. Taken together, our study sheds light on the dynamics of XCI progression and the asynchronicity between pluripotency, differentiation, and XCI.

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“…This technique does not require sophisticated platforms but is laborious and not very efficient. The laser capture microdissection (LCM) consists of coupling a laser system (infrared or ultraviolet) to a microscope that can capture a single cell from a solid tissue [116,117] ( Figure 3B). The advantages of LCM include visual inspection and spatial information of cells on the tissue and the possibility to store the remaining tissue.…”

Section: Single-cell Isolationmentioning

confidence: 99%

A Single Cell but Many Different Transcripts: A Journey into the World of Long Non-Coding RNAs

Alessio

Bonadio

Buson

et al. 2020

IJMS

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In late 2012 it was evidenced that most of the human genome is transcribed but only a small percentage of the transcripts are translated. This observation supported the importance of non-coding RNAs and it was confirmed in several organisms. The most abundant non-translated transcripts are long non-coding RNAs (lncRNAs). In contrast to protein-coding RNAs, they show a more cell-specific expression. To understand the function of lncRNAs, it is fundamental to investigate in which cells they are preferentially expressed and to detect their subcellular localization. Recent improvements of techniques that localize single RNA molecules in tissues like single-cell RNA sequencing and fluorescence amplification methods have given a considerable boost in the knowledge of the lncRNA functions. In recent years, single-cell transcription variability was associated with non-coding RNA expression, revealing this class of RNAs as important transcripts in the cell lineage specification. The purpose of this review is to collect updated information about lncRNA classification and new findings on their function derived from single-cell analysis. We also retained useful for all researchers to describe the methods available for single-cell analysis and the databases collecting single-cell and lncRNA data. Tables are included to schematize, describe, and compare exposed concepts.Int. J. Mol. Sci. 2020, 21, 302 2 of 32 not achieved by transcription factors (TFs) alone because of their low number (approximately 1500 in mammals [4]) compared to genes to regulate (90% of the genome is transcribed in human [5]). A single TF is estimated to regulate only ten thousand genes according to Chromatin immunoprecipitation and DNA sequencing experiments (ChIp-seq) [6]. Moreover, the involvement of non-coding RNAs in gene expression regulation was evident [7,8]. The evidence that the non-protein coding component of the human genome is actively transcribed and carries out crucial functions limits the importance of coding genes in genome regulation. Non-coding transcripts become one of the stars of modern biology, especially because of their involvement in a wide range of regulatory processes and changed the association of non-coding regions to junk DNA [3].The genome of multicellular eukaryotes is mostly comprised of non-coding DNA (less than 2% of the human genome codes for proteins [9] while 90% of the human genome is transcribed). Non-coding DNA is transcribed into different classes of non-coding RNAs (ncRNAs) that include structural RNAs (rRNAs and tRNAs) and regulatory RNAs [10]. rRNAs and tRNAs are involved in mRNA translation and can be long or short in size. Regulatory RNAs are further divided into three classes: Small, medium, and long non-coding RNAs. Small non-coding RNAs are between 20 and 50 nucleotides long and include microRNAs (miRNAs) that participate in post-transcriptional regulation [11], small interfering RNAs (siRNAs) that are double-stranded RNAs involved in gene silencing through RNA interfering pathway [12], piwi interacting R...

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Laser capture microscopy coupled with Smart-seq2 for precise spatial transcriptomic profiling

Cited by 276 publications

References 38 publications

Scaling single-cell genomics from phenomenology to mechanism

Scaling single-cell genomics from phenomenology to mechanism

Single-cell analyses of X Chromosome inactivation dynamics and pluripotency during differentiation

A Single Cell but Many Different Transcripts: A Journey into the World of Long Non-Coding RNAs

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