Determining mRNA Decay Rates Using RNA Approach to Equilibrium Sequencing (RATE-Seq).
Determining mRNA Decay Rates Using RNA Approach to Equilibrium Sequencing (RATE-Seq).
复制标题
DOI：
10.1007/978-1-4939-7540-2_2
复制
发表时间：
2018
影响因子：
--
通讯作者：
Farah Abdul-Rahman;D. Gresham
中科院分区：
文献类型：
--
作者：
Farah Abdul-Rahman;D. Gresham
RATE-seq是一种基于4-硫脲嘧啶（4-tU）的方法，能够在体内测量转录组范围的RNA降解速率。4-tU是一种尿嘧啶类似物，可快速掺入新合成的RNA中，并促进含有反应性巯基的生物素化分子的缀合。然后可以用链霉亲和素磁珠从未标记的RNA中分离生物素化的RNA。通过向在稳态条件下生长的细胞培养物中加入4-tU，在加入4-tU后的多个时间点分离标记的RNA群体，并使用RNAseq定量新转录的RNA的丰度，可以在单个实验中估计所有转录物的降解速率。RATE-seq数据的分析需要将RNAseq文库标准化为硫醇化RNA加标和非线性模型拟合以估计每种RNA种类的降解速率常数。
RATE-seq is a4-thiouracil (4-tU)-based method that enables the in vivo measurement of transcriptome-wide RNA degradation rates. 4-tU is an analog of uracil that is rapidly incorporated into newly synthesized RNA and facilitates the conjugation of a biotinylated molecule containing a reactive thiol group. The biotinylated RNA can then be fractionated from the unlabeled RNA with streptavidin magnetic beads. By adding 4-tU to a culture of cells growing in steady-state conditions, fractionating the labeled population of RNA at multiple time points following 4-tU addition, and quantifying the abundance of newly transcribed RNAs using RNAseq, it is possible to estimate the degradation rates of all transcripts in a single experiment. The analysis of the RATE-seq data entails normalization of RNAseq libraries to thiolated RNA spike-ins and nonlinear model fitting to estimate the degradation rate constant for each RNA species.