Isolation and RNA sequencing of single nuclei from Drosophila tissues.
Isolation and RNA sequencing of single nuclei from Drosophila tissues.
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果蝇组织中单核的分离及RNA测序。
DOI：
10.1016/j.xpro.2022.101417
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发表时间：
2022-06-17
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许多昆虫细胞被封装在外骨骼内，不能完整地解离，使得它们无法进行单细胞转录组学分析。我们已经使用单核RNA测序从多个果蝇组织中提取转录组信息。在这里，我们描述了（1）单个细胞核的解离，（2）使用两种流行的细胞分选仪分离单个细胞核，以及（3）为Smart-seq 2和10× Genomics制备文库的程序。该方案能够从单个细胞核生成高质量的转录组，并可应用于其他物种。有关本方案使用和执行的完整详细信息，请参见和。单核RNA-seq可以分析许多果蝇组织的转录组可以从新鲜或冷冻样品中提取高质量的细胞核细胞激活的细胞分选准确捕获果蝇细胞核可以使用10× Genomics或Smart-seq 2平台分析果蝇细胞核出版商说明：进行任何实验方案都需要遵守当地机构的实验室安全和道德准则。许多昆虫细胞被封装在外骨骼内，不能完整地解离，使得它们无法进行单细胞转录组学分析。我们已经使用单核RNA测序从多个果蝇组织中提取转录组信息。在这里，我们描述了（1）单个细胞核的解离，（2）使用两种流行的细胞分选仪分离单个细胞核，以及（3）为Smart-seq 2和10× Genomics制备文库的程序。该方案能够从单个细胞核生成高质量的转录组，并可应用于其他物种。
Many insect cells are encapsulated within the exoskeleton and cannot be dissociated intact, making them inaccessible to single-cell transcriptomic profiling. We have used single-nucleus RNA sequencing to extract transcriptomic information from multiple Drosophila tissues. Here, we describe procedures for the (1) dissociation of single nuclei, (2) isolation of single nuclei using two popular cell sorters, and (3) preparation of libraries for Smart-seq2 and 10× Genomics. This protocol enables generation of high-quality transcriptomes from single nuclei and can be applied to other species. For complete details on the use and execution of this protocol, please refer to and. Single-nucleus RNA-seq can profile transcriptomes from many Drosophila tissues High-quality nuclei can be extracted from fresh or frozen samples Fluorescence-activated cell sorting accurately captures Drosophila nuclei Fly nuclei can be profiled using 10× Genomics or Smart-seq2 platforms Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Many insect cells are encapsulated within the exoskeleton and cannot be dissociated intact, making them inaccessible to single-cell transcriptomic profiling. We have used single-nucleus RNA sequencing to extract transcriptomic information from multiple Drosophila tissues. Here, we describe procedures for the (1) dissociation of single nuclei, (2) isolation of single nuclei using two popular cell sorters, and (3) preparation of libraries for Smart-seq2 and 10× Genomics. This protocol enables generation of high-quality transcriptomes from single nuclei and can be applied to other species.