阿波贝克
计算生物学
5-羟甲基胞嘧啶
脱氨基
计算机科学
故障排除
工作流程
表观遗传学
管道(软件)
表观基因组
生物
遗传学
基因组
DNA甲基化
基因
生物化学
操作系统
数据库
基因表达
酶
程序设计语言
作者
Tong Wang,Meiqi Luo,Kiara N. Berríos,Emily K. Schutsky,Hao Wu,Rahul M. Kohli
出处
期刊:Methods in molecular biology
日期:2020-08-21
卷期号:: 349-367
被引量:12
标识
DOI:10.1007/978-1-0716-0876-0_27
摘要
Here, we provide a detailed protocol for our previously published technique, APOBEC-Coupled Epigenetic Sequencing (ACE-Seq), which localizes 5-hydroxymethylcytosine at single nucleotide resolution using nanogram quantities of input genomic DNA. In addition to describing suggested troubleshooting workflows, these methods include four important updates which should facilitate widespread implementation of the technique: (1) additionally optimized reaction conditions; (2) redesigned quality controls which can be performed prior to resource-consumptive deep sequencing; (3) confirmation that the less active, uncleaved APOBEC3A (A3A) fusion protein, which is easier to purify, can be used to perform ACE-Seq ; and (4) an example bioinformatic pipeline with suggested filtering strategies. Finally, we have provided a supplementary video which gives a narrated overview of the entire method and focuses on how best to perform the snap cool and A3A deamination steps central to successful execution of the method.
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