高通量测序 SELEX 用于体外测定 DNA 结合蛋白特异性。

High-throughput sequencing SELEX for the determination of DNA-binding protein specificities in vitro.

机构信息

The Wellcome Centre for Cell Biology, University of Edinburgh, Michael Swann Building, Max Born Crescent, The King's Buildings, Edinburgh EH9 3BF, UK.

The Wellcome Centre for Cell Biology, University of Edinburgh, Michael Swann Building, Max Born Crescent, The King's Buildings, Edinburgh EH9 3BF, UK; Informatics Forum, School of Informatics, University of Edinburgh, 10 Crichton Street, Edinburgh EH8 9AB, UK.

出版信息

STAR Protoc. 2022 Sep 16;3(3):101490. doi: 10.1016/j.xpro.2022.101490. Epub 2022 Jun 23.

DOI:10.1016/j.xpro.2022.101490

PMID:35776646

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9243297/

Abstract

High-throughput sequencing SELEX (HT-SELEX) is a powerful technique for unbiased determination of preferred target motifs of DNA-binding proteins in vitro. The procedure depends upon selection of DNA binding sites from a random library of oligonucleotides by purifying protein-DNA complexes and amplifying bound DNA using the polymerase chain reaction. Here, we describe an optimized step-by-step protocol for HT-SELEX compatible with Illumina sequencing. We also introduce a bioinformatic pipeline (eme_selex) facilitating the detection of promiscuous DNA binding by analyzing the enrichment of all possible k-mers. For complete details on the use and execution of this protocol, please refer to Pantier et al. (2021).

摘要

高通量测序 SELEX（HT-SELEX）是一种强大的技术，可在体外无偏地确定 DNA 结合蛋白的首选靶基序。该程序依赖于通过聚合酶链反应纯化蛋白-DNA 复合物并扩增结合 DNA 从随机寡核苷酸文库中选择 DNA 结合位点。在这里，我们描述了一种与 Illumina 测序兼容的优化分步协议。我们还介绍了一个生物信息学管道（eme_selex），通过分析所有可能的 k-mer 的富集来促进对混杂 DNA 结合的检测。有关此协议的使用和执行的完整详细信息，请参阅 Pantier 等人。（2021 年）。