Basnet Harihar, Massague Joan
Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA.
Bio Protoc. 2019 Nov 20;9(22):e3433. doi: 10.21769/BioProtoc.3433.
Tissues are comprised of different cell types whose interactions elicit distinct gene expression patterns that regulate tissue formation, regeneration, homeostasis and repair. Analysis of these gene expression patterns require methods that can capture as closely as possible the transcriptomes of cells of interest in their tissue microenvironment. Current technologies designed to study transcriptomics are limited by their low sensitivity that require cell types to represent more than 1% of the total tissue, making it challenging to transcriptionally profile rare cell populations rapidly isolated from their native microenvironment. To address this problem, we developed fluorouracil-tagged RNA sequencing (Flura-seq) that utilizes cytosine deaminase (CD) to convert the non-natural pyrimidine fluorocytosine to fluorouracil. Expression of CD and exposure to fluorocytosine generates fluorouracil and metabolically labels newly synthesized RNAs specifically in cells of interest. Fluorouracil-tagged RNAs can then be immunopurified and used for downstream analysis. Here, we describe the detailed protocol to perform Flura-seq both and . The robustness, simplicity and lack of toxicity of Flura-seq make this tool broadly applicable to many studies in developmental, regenerative, and cancer biology.
组织由不同的细胞类型组成，这些细胞类型之间的相互作用引发独特的基因表达模式，从而调节组织的形成、再生、稳态和修复。对这些基因表达模式的分析需要能够尽可能紧密地捕捉目标细胞在其组织微环境中的转录组的方法。目前用于研究转录组学的技术受到其低灵敏度的限制，这种低灵敏度要求细胞类型在总组织中所占比例超过1%，这使得对从其天然微环境中快速分离出的稀有细胞群体进行转录谱分析具有挑战性。为了解决这个问题，我们开发了氟尿嘧啶标记的RNA测序（Flura-seq），该方法利用胞嘧啶脱氨酶（CD）将非天然嘧啶氟胞嘧啶转化为氟尿嘧啶。CD的表达和氟胞嘧啶的暴露会产生氟尿嘧啶，并特异性地在目标细胞中对新合成的RNA进行代谢标记。然后可以对氟尿嘧啶标记的RNA进行免疫纯化，并用于下游分析。在这里，我们描述了进行Flura-seq的详细方案。Flura-seq的稳健性、简单性和无毒性使其成为一种广泛适用于发育生物学、再生生物学和癌症生物学许多研究的工具。
