{"doi":"10.21769/bioprotoc.3749","title":"Probe-Seq: Method for RNA Sequencing of Specific Cell Types from Animal Tissue","abstract":"Most organs and tissues are composed of many types of cells. To characterize cellular state, various transcription profiling approaches are currently available, including whole-tissue bulk RNA sequencing, single cell RNA sequencing (scRNA-Seq), and cell type-specific RNA sequencing. What is missing in this repertoire is a simple, versatile method for bulk transcriptional profiling of cell types for which cell type-specific genetic markers or antibodies are not readily available. We therefore developed Probe-Seq, which uses hybridization of gene-specific probes to RNA markers for isolation of specific types of cells, to enable downstream FACS isolation and bulk RNA sequencing. We show that this method can enable isolation and profiling of specific cell types from mouse retina, frozen human retina, Drosophila midgut, and developing chick retina, suggesting that it is likely useful for most organisms., [摘要 ] 大多数器官和组织由许多类型的细胞组成。为了表征细胞状态，目前可以使用各种转录分析方法，包括全组织体RNA测序，单细胞RNA测序（scRNA -Seq）和特定于细胞类型的RNA测序。在此库中缺少的是一种简单，通用的方法，无法批量获得细胞类型的特定基因标记或抗体，因此无法批量转录。因此，我们开发了Probe-Seq，该探针使用基因特异性探针与RNA标记的杂交来分离特定类型的细胞，以实现下游FACS分离和大量RNA测序。我们表明，该方法可以实现从小鼠视网膜，冷冻人视网膜，果蝇中肠和发育中的雏鸡视网膜中分离和分析特定细胞类型的特征，这表明它对大多数生物很有用。[背景技术 [ 0002 ] 在过去的二十年中，使用RNA-Seq和微阵列进行转录谱分析已在生物学研究中无处不在。分析现在是用来了解大多数生物体中细胞和细胞状态的主要工具之一。它被用于正常发育，异常发育和疾病的研究，并极大地扩展了我们对进化关系的理解。特别地，scRNA- Seq已经以前所未有的速度导致了新型细胞类型的鉴定（Picelli 等，2013；Jaitin 等，2014； Klein 等，2015； Macosko 等，2015）。为了更深入地了解这些新描述的细胞类型，一种无需转基因标记或特异性抗原即可将其分离的方法将大有裨益。尽管可以使用scRNA -Seq来获得所有细胞类型的更深覆盖，但是这种方法需要对大量细胞类型进行大量测序，甚至可以对不那么丰富的细胞类型进行适度的覆盖。 为了补充单细胞形态，我们开发了Probe-Seq，利用scRNA -Seq努力定义的最新可用的RNA标记，可以从任何生物体进行任何细胞类型的转录概况分析。Probe-Seq使用专为SABER荧光原位杂交（FISH）设计的基因特异性探针组（Kishi 等，2019）对离解的固定细胞或细胞核中的特定RNA标记进行荧光标记，以进行下游FACS分离和RNA测序。尽管FISH先前已用于体外培养的细胞的FACS和RNA测序（Klemm et al。，2014），但Probe-Seq可直接从组织中提取细胞，如我们的小鼠视网膜，人冷冻视网膜，果蝇中肠所示，并发育小鸡视网膜。迄今为止，它已与所有测试过的生物和组织合作，表明它可能对任何生物都有用（Amamoto 等人，2019）。该方法也是成本和时间有效的（图1）。Probe-Seq可以在不到三天的时间内，不到6个小时的动手时间就可以完成准备测序的文库。而且，整个过程不需要任何专用设备，并且大多数试剂可以在常规分子生物学实验室中找到。图1 。FL 流图表对探针- SEQ协议","journal":"BIO-PROTOCOL","year":2020,"id":116926,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9279,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2020-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":363703,"name":"M Garcia","orcid":"0000-0003-2418-541X","position":1,"is_corresponding":false},{"id":545849,"name":"Emma R. West","orcid":"0000-0002-2109-016X","position":2,"is_corresponding":false},{"id":545850,"name":"Jiho Choi","orcid":"0000-0001-8839-4056","position":3,"is_corresponding":false},{"id":546408,"name":"Sylvain W. Lapan","orcid":null,"position":4,"is_corresponding":false},{"id":265246,"name":"Elizabeth A. Lane","orcid":null,"position":5,"is_corresponding":false},{"id":14957,"name":"Norbert Perrimon","orcid":"0000-0001-7542-472X","position":6,"is_corresponding":false},{"id":247143,"name":"Constance L. Cepko","orcid":"0000-0002-9945-6387","position":7,"is_corresponding":false},{"id":545848,"name":"Ryoji Amamoto","orcid":"0000-0002-9335-112X","position":0,"is_corresponding":true}],"reference_count":12,"raw_metadata":null,"created_at":"2026-07-18T23:13:47.803267Z","pmid":"33659409","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}