Thiol-linked alkylation of RNA to assess expression dynamics.
Thiol-linked alkylation of RNA to assess expression dynamics.
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DOI：
10.1038/nmeth.4435
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发表时间：
2017-12
期刊：
影响因子：
48
通讯作者：
Ameres SL
中科院分区：
文献类型：
--
作者：
Herzog VA;Reichholf B;Neumann T;Rescheneder P;Bhat P;Burkard TR;Wlotzka W;von Haeseler A;Zuber J;Ameres SL
高通量测序的基因表达谱揭示了稳定状态下RNA物种的定性和定量变化，但掩盖了RNA转录、加工和衰退的细胞内动力学。我们开发了用于RNA代谢测序的硫醇(SH)连接烷基化(SLAM-SEQ)，这是一种基于正交化学的RNA测序技术，以单核苷酸分辨率检测RNA物种中4-硫代尿苷(S4U)的掺入。SLAM-SEQ与成熟的代谢RNA标记方案相结合，并结合标准的、低输入、高通量的RNA测序方法，使人们能够在总RNA的背景下快速获得依赖于RNA聚合酶II的基因表达动态。我们在小鼠胚胎干细胞中验证了该方法，表明依赖RNA聚合酶II的转录输出与Oct4/Sox2/Nanog定义的增强子活性成比例；我们为由microRNAs和N6-甲基腺苷启动的转录后基因调控途径介导的转录特异性RNA周转提供了定量和机械证据。SLAM-SEQ促进了对以可访问的、经济有效的和可扩展的方式控制基因表达的基本机制的剖析。
Gene expression profiling by high-throughput sequencing reveals qualitative and quantitative changes in RNA species at steady-state but obscures the intracellular dynamics of RNA transcription, processing and decay. We developed thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq), an orthogonal chemistry-based RNA sequencing technology that detects 4-thiouridine (s4U)-incorporation in RNA species at single-nucleotide resolution. In combination with well-established metabolic RNA labeling protocols and coupled to standard, low-input, high-throughput RNA sequencing methods, SLAM-seq enables rapid access to RNA polymerase II-dependent gene expression dynamics in the context of total RNA. We validated the method in mouse embryonic stem cells by showing that the RNA-polymerase II-dependent transcriptional output scales with Oct4/Sox2/Nanog-defined enhancer activity; and we provide quantitative and mechanistic evidence for transcript-specific RNA turnover mediated by post-transcriptional gene regulatory pathways initiated by microRNAs and N6-methyladenosine. SLAM-seq facilitates the dissection of fundamental mechanisms that control gene expression in an accessible, cost-effective, and scalable manner.
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DOI：
10.1093/bioinformatics/btv308
发表时间：
2015-09-15
期刊：
Bioinformatics (Oxford, England)
影响因子：
--
作者：
Hodgkins A;Farne A;Perera S;Grego T;Parry-Smith DJ;Skarnes WC;Iyer V
通讯作者：
Iyer V
使用有效和可逆的共价化学跟踪不同的RNA群体。
影响因子：
16
作者：
Duffy EE;Rutenberg-Schoenberg M;Stark CD;Kitchen RR;Gerstein MB;Simon MD
通讯作者：
Simon MD
影响因子：
64.5
作者：
Hafner M;Landthaler M;Burger L;Khorshid M;Hausser J;Berninger P;Rothballer A;Ascano M Jr;Jungkamp AC;Munschauer M;Ulrich A;Wardle GS;Dewell S;Zavolan M;Tuschl T
通讯作者：
Tuschl T
影响因子：
23.9
作者：
Batista, Pedro J.;Molinie, Benoit;Wang, Jinkai;Qu, Kun;Zhang, Jiajing;Li, Lingjie;Bouley, Donna M.;Lujan, Ernesto;Haddad, Bahareh;Daneshvar, Kaveh;Carter, Ava C.;Flynn, Ryan A.;Zhou, Chan;Lim, Kok-Seong;Dedon, Peter;Wernig, Marius;Mullen, Alan C.;Xing, Yi;Giallourakis, Cosmas C.;Chang, Howard Y.
通讯作者：
Chang, Howard Y.
影响因子：
4.1
作者：
Burger, Kaspar;Muehl, Bastian;Eick, Dirk
通讯作者：
Eick, Dirk