Nature Protocols最新文献_第2页

Molecular recording using DNA Typewriter. 使用 DNA 打字机进行分子记录。

IF 14.8 1区生物学

Nature Protocols Pub Date : 2024-06-06 DOI: 10.1038/s41596-024-01003-0

Hanna Liao, Junhong Choi, Jay Shendure

{"title":"Molecular recording using DNA Typewriter.","authors":"Hanna Liao, Junhong Choi, Jay Shendure","doi":"10.1038/s41596-024-01003-0","DOIUrl":"https://doi.org/10.1038/s41596-024-01003-0","url":null,"abstract":"Recording molecular information to genomic DNA is a powerful means of investigating topics ranging from multicellular development to cancer evolution. With molecular recording based on genome editing, events such as cell divisions and signaling pathway activity drive specific alterations in a cell's DNA, marking the genome with information about a cell's history that can be read out after the fact. Although genome editing has been used for molecular recording, capturing the temporal relationships among recorded events in mammalian cells remains challenging. The DNA Typewriter system overcomes this limitation by leveraging prime editing to facilitate sequential insertions to an engineered genomic region. DNA Typewriter includes three distinct components: DNA Tape as the 'substrate' to which edits accrue in an ordered manner, the prime editor enzyme, and prime editing guide RNAs, which program insertional edits to DNA Tape. In this protocol, we describe general design considerations for DNA Typewriter, step-by-step instructions on how to perform recording experiments by using DNA Typewriter in HEK293T cells, and example scripts for analyzing DNA Typewriter data ( https://doi.org/10.6084/m9.figshare.22728758 ). This protocol covers two main applications of DNA Typewriter: recording sequential transfection events with programmed barcode insertions by using prime editing and recording lineage information during the expansion of a single cell to many. Compared with other methods that are compatible with mammalian cells, DNA Typewriter enables the recording of temporal information with higher recording capacities and can be completed within 4-6 weeks with basic expertise in molecular cloning, mammalian cell culturing and DNA sequencing data analysis.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":14.8,"publicationDate":"2024-06-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141284272","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Scanorama: integrating large and diverse single-cell transcriptomic datasets. Scanorama：整合大型多样的单细胞转录组数据集。

IF 14.8 1区生物学

Nature Protocols Pub Date : 2024-06-06 DOI: 10.1038/s41596-024-00991-3

Brian L Hie, Soochi Kim, Thomas A Rando, Bryan Bryson, Bonnie Berger

{"title":"Scanorama: integrating large and diverse single-cell transcriptomic datasets.","authors":"Brian L Hie, Soochi Kim, Thomas A Rando, Bryan Bryson, Bonnie Berger","doi":"10.1038/s41596-024-00991-3","DOIUrl":"https://doi.org/10.1038/s41596-024-00991-3","url":null,"abstract":"Merging diverse single-cell RNA sequencing (scRNA-seq) data from numerous experiments, laboratories and technologies can uncover important biological insights. Nonetheless, integrating scRNA-seq data encounters special challenges when the datasets are composed of diverse cell type compositions. Scanorama offers a robust solution for improving the quality and interpretation of heterogeneous scRNA-seq data by effectively merging information from diverse sources. Scanorama is designed to address the technical variation introduced by differences in sample preparation, sequencing depth and experimental batches that can confound the analysis of multiple scRNA-seq datasets. Here we provide a detailed protocol for using Scanorama within a Scanpy-based single-cell analysis workflow coupled with Google Colaboratory, a cloud-based free Jupyter notebook environment service. The protocol involves Scanorama integration, a process that typically spans 0.5-3 h. Scanorama integration requires a basic understanding of cellular biology, transcriptomic technologies and bioinformatics. Our protocol and new Scanorama-Colaboratory resource should make scRNA-seq integration more widely accessible to researchers.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":14.8,"publicationDate":"2024-06-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141284273","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

All-optical voltage imaging-guided postsynaptic single-cell transcriptome profiling with Voltage-Seq. 利用 Voltage-Seq 进行全光学电压成像引导的突触后单细胞转录组分析。

IF 14.8 1区生物学

Nature Protocols Pub Date : 2024-06-04 DOI: 10.1038/s41596-024-01005-y

Veronika Csillag, J C Noble, Daniela Calvigioni, Björn Reinius, János Fuzik

{"title":"All-optical voltage imaging-guided postsynaptic single-cell transcriptome profiling with Voltage-Seq.","authors":"Veronika Csillag, J C Noble, Daniela Calvigioni, Björn Reinius, János Fuzik","doi":"10.1038/s41596-024-01005-y","DOIUrl":"https://doi.org/10.1038/s41596-024-01005-y","url":null,"abstract":"Neuronal pathways recruit large postsynaptic populations and maintain connections via distinct postsynaptic response types (PRTs). Until recently, PRTs were accessible as a selection criterion for single-cell RNA sequencing only through probing by low-throughput whole-cell electrophysiology. To overcome these limitations and target neurons on the basis of specific PRTs for soma collection and subsequent single-cell RNA sequencing, we developed Voltage-Seq using the genetically encoded voltage indicator Voltron in acute brain slices from mice. We also created an onsite analysis tool, VoltView, to guide soma collection of specific PRTs using a classifier based on a previously acquired database of connectomes from multiple animals. Here we present our procedure for preparing the optical path, the imaging setup and detailing the imaging and analysis steps, as well as a complete procedure for sequencing library preparation. This enables researchers to conduct our high-throughput all-optical synaptic assay and to obtain single-cell transcriptomic data from selected postsynaptic neurons. This also allows researchers to resolve the connectivity ratio of a specific pathway and explore the diversity of PRTs within that connectome. Furthermore, combining high throughput with quick analysis gives unique access to find specific connections within a large postsynaptic connectome. Voltage-Seq also allows the investigation of correlations between connectivity and gene expression changes in a postsynaptic cell-type-specific manner for both excitatory and inhibitory connections. The Voltage-Seq workflow can be completed in ~6 weeks, including 4-5 weeks for viral expression of the Voltron sensor. The technique requires knowledge of basic laboratory techniques, micromanipulator handling skills and experience in molecular biology and bioinformatics.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":14.8,"publicationDate":"2024-06-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141248028","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Author Correction: CRISPR-Cas9-based genome-wide screening of Toxoplasma gondii. 作者更正：基于 CRISPR-Cas9 的弓形虫全基因组筛选。

IF 14.8 1区生物学

Nature Protocols Pub Date : 2024-06-04 DOI: 10.1038/s41596-024-01018-7

Saima M Sidik, Diego Huet, Sebastian Lourido

引用次数: 0

Real-time imaging of axonal membrane protein life cycles. 轴突膜蛋白生命周期的实时成像。

IF 14.8 1区生物学

Nature Protocols Pub Date : 2024-06-03 DOI: 10.1038/s41596-024-00997-x

Sidharth Tyagi, Grant P Higerd-Rusli, Elizabeth J Akin, Christopher A Baker, Shujun Liu, Fadia B Dib-Hajj, Stephen G Waxman, Sulayman D Dib-Hajj

{"title":"Real-time imaging of axonal membrane protein life cycles.","authors":"Sidharth Tyagi, Grant P Higerd-Rusli, Elizabeth J Akin, Christopher A Baker, Shujun Liu, Fadia B Dib-Hajj, Stephen G Waxman, Sulayman D Dib-Hajj","doi":"10.1038/s41596-024-00997-x","DOIUrl":"10.1038/s41596-024-00997-x","url":null,"abstract":"The construction of neuronal membranes is a dynamic process involving the biogenesis, vesicular packaging, transport, insertion and recycling of membrane proteins. Optical imaging is well suited for the study of protein spatial organization and transport. However, various shortcomings of existing imaging techniques have prevented the study of specific types of proteins and cellular processes. Here we describe strategies for protein tagging and labeling, cell culture and microscopy that enable the real-time imaging of axonal membrane protein trafficking and subcellular distribution as they progress through some stages of their life cycle. First, we describe a process for engineering membrane proteins with extracellular self-labeling tags (either HaloTag or SNAPTag), which can be labeled with fluorescent ligands of various colors and cell permeability, providing flexibility for investigating the trafficking and spatiotemporal regulation of multiple membrane proteins in neuronal compartments. Next, we detail the dissection, transfection and culture of dorsal root ganglion sensory neurons in microfluidic chambers, which physically compartmentalizes cell bodies and distal axons. Finally, we describe four labeling and imaging procedures that utilize these enzymatically tagged proteins, flexible fluorescent labels and compartmentalized neuronal cultures to study axonal membrane protein anterograde and retrograde transport, the cotransport of multiple proteins, protein subcellular localization, exocytosis and endocytosis. Additionally, we generated open-source software for analyzing the imaging data in a high throughput manner. The experimental and analysis workflows provide an approach for studying the dynamics of neuronal membrane protein homeostasis, addressing longstanding challenges in this area. The protocol requires 5-7 days and expertise in cell culture and microscopy.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":14.8,"publicationDate":"2024-06-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141238035","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Integrating genome-wide CRISPR screens and in silico drug profiling for targeted antidote development. 整合全基因组 CRISPR 筛选和硅学药物分析，开发靶向解毒剂。

IF 14.8 1区生物学

Nature Protocols Pub Date : 2024-05-30 DOI: 10.1038/s41596-024-00995-z

Bei Wang, Yu Xu, Arabella H Wan, Guohui Wan, Qiao-Ping Wang

{"title":"Integrating genome-wide CRISPR screens and in silico drug profiling for targeted antidote development.","authors":"Bei Wang, Yu Xu, Arabella H Wan, Guohui Wan, Qiao-Ping Wang","doi":"10.1038/s41596-024-00995-z","DOIUrl":"https://doi.org/10.1038/s41596-024-00995-z","url":null,"abstract":"Numerous toxins threaten humans, but specific antidotes are unavailable for most of them. Although CRISPR screening has aided the discovery of the mechanisms of some toxins, developing targeted antidotes remains a significant challenge. Recently, we established a systematic framework to develop antidotes by combining the identification of novel drug targets by using a genome-wide CRISPR screen with a virtual screen of drugs approved by the US Food and Drug Administration. This approach allows for a comprehensive understanding of toxin mechanisms at the whole-genome level and facilitates the identification of promising antidote drugs targeting specific molecules. Here, we present step-by-step instructions for executing genome-scale CRISPR-Cas9 knockout screens of toxins in HAP1 cells. We also provide detailed guidance for conducting an in silico drug screen and an in vivo drug validation. By using this protocol, it takes ~4 weeks to perform the genome-scale screen, 4 weeks for sequencing and data analysis, 4 weeks to validate candidate genes, 1 week for the virtual screen and 2 weeks for in vitro drug validation. This framework has the potential to accelerate the development of antidotes for a wide range of toxins and can rapidly identify promising drug candidates that are already known to be safe and effective. This could lead to the development of new antidotes much more quickly than traditional methods, protecting lives from diverse toxins and advancing human health.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":14.8,"publicationDate":"2024-05-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141179179","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Morphological and molecular preservation through universal preparation of fresh-frozen tissue samples for multimodal imaging workflows. 通过为多模态成像工作流程普遍制备新鲜冷冻组织样本，实现形态和分子保存。

IF 14.8 1区生物学

Nature Protocols Pub Date : 2024-05-28 DOI: 10.1038/s41596-024-00987-z

Andreas Dannhorn, Emine Kazanc, Lucy Flint, Fei Guo, Alfie Carter, Andrew R Hall, Stewart A Jones, George Poulogiannis, Simon T Barry, Owen J Sansom, Josephine Bunch, Zoltan Takats, Richard J A Goodwin

{"title":"Morphological and molecular preservation through universal preparation of fresh-frozen tissue samples for multimodal imaging workflows.","authors":"Andreas Dannhorn, Emine Kazanc, Lucy Flint, Fei Guo, Alfie Carter, Andrew R Hall, Stewart A Jones, George Poulogiannis, Simon T Barry, Owen J Sansom, Josephine Bunch, Zoltan Takats, Richard J A Goodwin","doi":"10.1038/s41596-024-00987-z","DOIUrl":"https://doi.org/10.1038/s41596-024-00987-z","url":null,"abstract":"The landscape of tissue-based imaging modalities is constantly and rapidly evolving. While formalin-fixed, paraffin-embedded material is still useful for histological imaging, the fixation process irreversibly changes the molecular composition of the sample. Therefore, many imaging approaches require fresh-frozen material to get meaningful results. This is particularly true for molecular imaging techniques such as mass spectrometry imaging, which are widely used to probe the spatial arrangement of the tissue metabolome. As high-quality fresh-frozen tissues are limited in their availability, any sample preparation workflow they are subjected to needs to ensure morphological and molecular preservation of the tissues and be compatible with as many of the established and emerging imaging techniques as possible to obtain the maximum possible insights from the tissues. Here we describe a universal sample preparation workflow, from the initial step of freezing the tissues to the cold embedding in a new hydroxypropyl methylcellulose/polyvinylpyrrolidone-enriched hydrogel and the generation of thin tissue sections for analysis. Moreover, we highlight the optimized storage conditions that limit molecular and morphological degradation of the sections. The protocol is compatible with human and plant tissues and can be easily adapted for the preparation of alternative sample formats (e.g., three-dimensional cell cultures). The integrated workflow is universally compatible with histological tissue analysis, mass spectrometry imaging and imaging mass cytometry, as well as spatial proteomic, genomic and transcriptomic tissue analysis. The protocol can be completed within 4 h and requires minimal prior experience in the preparation of tissue samples for multimodal imaging experiments.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":14.8,"publicationDate":"2024-05-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141162099","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

An MSstats workflow for detecting differentially abundant proteins in large-scale data-independent acquisition mass spectrometry experiments with FragPipe processing. 利用 FragPipe 处理在大规模数据独立采集质谱实验中检测差异丰富蛋白质的 MSstats 工作流程。

IF 14.8 1区生物学

Nature Protocols Pub Date : 2024-05-20 DOI: 10.1038/s41596-024-01000-3

Devon Kohler, Mateusz Staniak, Fengchao Yu, Alexey I Nesvizhskii, Olga Vitek

{"title":"An MSstats workflow for detecting differentially abundant proteins in large-scale data-independent acquisition mass spectrometry experiments with FragPipe processing.","authors":"Devon Kohler, Mateusz Staniak, Fengchao Yu, Alexey I Nesvizhskii, Olga Vitek","doi":"10.1038/s41596-024-01000-3","DOIUrl":"https://doi.org/10.1038/s41596-024-01000-3","url":null,"abstract":"Technological advances in mass spectrometry and proteomics have made it possible to perform larger-scale and more-complex experiments. The volume and complexity of the resulting data create major challenges for downstream analysis. In particular, next-generation data-independent acquisition (DIA) experiments enable wider proteome coverage than more traditional targeted approaches but require computational workflows that can manage much larger datasets and identify peptide sequences from complex and overlapping spectral features. Data-processing tools such as FragPipe, DIA-NN and Spectronaut have undergone substantial improvements to process spectral features in a reasonable time. Statistical analysis tools are needed to draw meaningful comparisons between experimental samples, but these tools were also originally designed with smaller datasets in mind. This protocol describes an updated version of MSstats that has been adapted to be compatible with large-scale DIA experiments. A very large DIA experiment, processed with FragPipe, is used as an example to demonstrate different MSstats workflows. The choice of workflow depends on the user's computational resources. For datasets that are too large to fit into a standard computer's memory, we demonstrate the use of MSstatsBig, a companion R package to MSstats. The protocol also highlights key decisions that have a major effect on both the results and the processing time of the analysis. The MSstats processing can be expected to take 1-3 h depending on the usage of MSstatsBig. The protocol can be run in the point-and-click graphical user interface MSstatsShiny or implemented with minimal coding expertise in R.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":14.8,"publicationDate":"2024-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141071601","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Tutorial: design, production and testing of oncolytic viruses for cancer immunotherapy. 教程：用于癌症免疫疗法的溶瘤病毒的设计、生产和测试。

IF 14.8 1区生物学

Nature Protocols Pub Date : 2024-05-20 DOI: 10.1038/s41596-024-00985-1

Shashi Gujar, Jonathan G Pol, Vishnupriyan Kumar, Manuela Lizarralde-Guerrero, Prathyusha Konda, Guido Kroemer, John C Bell

{"title":"Tutorial: design, production and testing of oncolytic viruses for cancer immunotherapy.","authors":"Shashi Gujar, Jonathan G Pol, Vishnupriyan Kumar, Manuela Lizarralde-Guerrero, Prathyusha Konda, Guido Kroemer, John C Bell","doi":"10.1038/s41596-024-00985-1","DOIUrl":"https://doi.org/10.1038/s41596-024-00985-1","url":null,"abstract":"Oncolytic viruses (OVs) represent a novel class of cancer immunotherapy agents that preferentially infect and kill cancer cells and promote protective antitumor immunity. Furthermore, OVs can be used in combination with established or upcoming immunotherapeutic agents, especially immune checkpoint inhibitors, to efficiently target a wide range of malignancies. The development of OV-based therapy involves three major steps before clinical evaluation: design, production and preclinical testing. OVs can be designed as natural or engineered strains and subsequently selected for their ability to kill a broad spectrum of cancer cells rather than normal, healthy cells. OV selection is further influenced by multiple factors, such as the availability of a specific viral platform, cancer cell permissivity, the need for genetic engineering to render the virus non-pathogenic and/or more effective and logistical considerations around the use of OVs within the laboratory or clinical setting. Selected OVs are then produced and tested for their anticancer potential by using syngeneic, xenograft or humanized preclinical models wherein immunocompromised and immunocompetent setups are used to elucidate their direct oncolytic ability as well as indirect immunotherapeutic potential in vivo. Finally, OVs demonstrating the desired anticancer potential progress toward translation in patients with cancer. This tutorial provides guidelines for the design, production and preclinical testing of OVs, emphasizing considerations specific to OV technology that determine their clinical utility as cancer immunotherapy agents.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":14.8,"publicationDate":"2024-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141071553","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

ProBac-seq, a bacterial single-cell RNA sequencing methodology using droplet microfluidics and large oligonucleotide probe sets. ProBac-seq 是一种使用液滴微流控技术和大型寡核苷酸探针组的细菌单细胞 RNA 测序方法。

IF 14.8 1区生物学

Nature Protocols Pub Date : 2024-05-20 DOI: 10.1038/s41596-024-01002-1

Prosenjit Samanta, Samuel F Cooke, Ryan McNulty, Sahand Hormoz, Adam Rosenthal

{"title":"ProBac-seq, a bacterial single-cell RNA sequencing methodology using droplet microfluidics and large oligonucleotide probe sets.","authors":"Prosenjit Samanta, Samuel F Cooke, Ryan McNulty, Sahand Hormoz, Adam Rosenthal","doi":"10.1038/s41596-024-01002-1","DOIUrl":"https://doi.org/10.1038/s41596-024-01002-1","url":null,"abstract":"Methods that measure the transcriptomic state of thousands of individual cells have transformed our understanding of cellular heterogeneity in eukaryotic cells since their introduction in the past decade. While simple and accessible protocols and commercial products are now available for the processing of mammalian cells, these existing technologies are incompatible with use in bacterial samples for several fundamental reasons including the absence of polyadenylation on bacterial messenger RNA, the instability of bacterial transcripts and the incompatibility of bacterial cell morphology with existing methodologies. Recently, we developed ProBac sequencing (ProBac-seq), a method that overcomes these technical difficulties and provides high-quality single-cell gene expression data from thousands of bacterial cells by using messenger RNA-specific probes. Here we provide details for designing large oligonucleotide probe sets for an organism of choice, amplifying probe sets to produce sufficient quantities for repeated experiments, adding unique molecular indexes and poly-A tails to produce finalized probes, in situ probe hybridization and single-cell encapsulation and library preparation. This protocol, from the probe amplification to the library preparation, requires ~7 d to complete. ProBac-seq offers several advantages over other methods by capturing only the desired target sequences and avoiding nondesired transcripts, such as highly abundant ribosomal RNA, thus enriching for signal that better informs on cellular state. The use of multiple probes per gene can detect meaningful single-cell signals from cells expressing transcripts to a lesser degree or those grown in minimal media and other environmentally relevant conditions in which cells are less active. ProBac-seq is also compatible with other organisms that can be profiled by in situ hybridization techniques.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":null,"pages":null},"PeriodicalIF":14.8,"publicationDate":"2024-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141071605","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0