Cell cycle specific, differentially tagged ribosomal proteins to measure phase specific transcriptomes from asynchronously cycling cells.
Cell cycle specific, differentially tagged ribosomal proteins to measure phase specific transcriptomes from asynchronously cycling cells.
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细胞周期特异性的、带有差异标记的核糖体蛋白，用于测量来自异步循环细胞的阶段特异性转录组。
DOI：
10.1038/s41598-024-52085-5
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发表时间：
2024-01-18
影响因子：
4.6
通讯作者：
中科院分区：
文献类型：
--
作者：
异步循环细胞对阶段特异性基因表达的准确表征构成了挑战。当前的策略，包括RNA测序，是对细胞周期内的稳态基因表达进行检测，并且由于无法解析动态基因调控网络而存在固有局限性。单细胞RNA测序（scRNAseq）如果存在足够多的循环细胞，能够识别不同的细胞周期转录组，然而有些细胞不适合进行scRNA测序。因此，我们融合了两种强大的策略，即荧光泛素化细胞周期指示剂（FUCCI）细胞周期传感器中使用的CDT1和GMNN降解决定子，以及RiboTrap/Tag技术中使用的核糖体蛋白表位标记，以分离细胞周期阶段特异性mRNA用于测序。由此产生的细胞周期依赖性标记核糖体蛋白（ccTaggedRP）在细胞周期中差异表达，与内源性核糖体蛋白具有相似的亚细胞定位，整合到核糖体和多聚核糖体中，并有助于回收细胞周期阶段特异性RNA用于测序。ccTaggedRP在研究复杂细胞群体中的阶段特异性基因表达方面具有广泛的应用。
Asynchronously cycling cells pose a challenge to the accurate characterization of phase-specific gene expression. Current strategies, including RNAseq, survey the steady state gene expression across the cell cycle and are inherently limited by their inability to resolve dynamic gene regulatory networks. Single cell RNAseq (scRNAseq) can identify different cell cycle transcriptomes if enough cycling cells are present, however some cells are not amenable to scRNAseq. Therefore, we merged two powerful strategies, the CDT1 and GMNN degrons used in Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) cell cycle sensors and the ribosomal protein epitope tagging used in RiboTrap/Tag technologies to isolate cell cycle phase-specific mRNA for sequencing. The resulting cell cycle dependent, tagged ribosomal proteins (ccTaggedRP) were differentially expressed during the cell cycle, had similar subcellular locations as endogenous ribosomal proteins, incorporated into ribosomes and polysomes, and facilitated the recovery of cell cycle phase-specific RNA for sequencing. ccTaggedRP has broad applications to investigate phase-specific gene expression in complex cell populations.
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影响因子：
14.9
作者：
Fischer M;Grossmann P;Padi M;DeCaprio JA
通讯作者：
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64.8
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Teichmann SA
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Hanson, Jesse E.
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Li, Guang;Xu, Adele;Wu, Sean M.
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Wu, Sean M.
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通讯作者：
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