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

Raphaël Pantier; Kashyap Chhatbar; Grace Alston; Heng Yang Lee; Adrian Bird

doi:10.1016/j.xpro.2022.101490

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

Raphaël Pantier, Kashyap Chhatbar, Grace Alston, Heng Yang Lee, Adrian Bird

Research output: Contribution to journal › Article › peer-review

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).

Original language	English
Article number	101490
Number of pages	21
Journal	Star Protocols
Volume	3
Issue number	3
Early online date	22 Jun 2022
DOIs	https://doi.org/10.1016/j.xpro.2022.101490
Publication status	Published - 16 Sept 2022

Keywords / Materials (for Non-textual outputs)

bioinformatics
high throughput screening
molecular biology
sequence analysis
sequencing

Access to Document

10.1016/j.xpro.2022.101490Licence: Creative Commons: Attribution (CC-BY)

BirdEtallSTAR2022HighThroughputSequencingSELEXForTheDeterminationFinal published version, 3.37 MBLicence: Creative Commons: Attribution (CC-BY)

Core funding for the Wellcome Centre for Cell Biology
Marston, A. (Principal Investigator)
UK-based charities
1/12/21 → 30/11/23
Project: Research
Wellcome Centre for Cell Biology
Tollervey, D. (Principal Investigator)
UK-based charities
1/12/16 → 1/12/21
Project: Research
Towards understanding and treatment of MeCP2-related disorders
Bird, A. (Principal Investigator)
UK-based charities
1/01/16 → 30/06/22
Project: Research

Cite this

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title = "High-throughput sequencing SELEX for the determination of DNA-binding protein specificities in vitro",

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).",

keywords = "bioinformatics, high throughput screening, molecular biology, sequence analysis, sequencing",

author = "Rapha{\"e}l Pantier and Kashyap Chhatbar and Grace Alston and Lee, {Heng Yang} and Adrian Bird",

note = "Funding Information: We thank Arttu Jolma and Jussi Taipale for useful advice regarding HT-SELEX library design. We thank Vladimir Benes and the EMBL Genecore Facility for technical support with high-throughput sequencing and library design. This work was funded by European Research Council Advanced Grant EC 694295 Gen-Epix and Wellcome Investigator Award 107930 to A.B., who is also a member of the Simons Initiative for the Developing Brain. The Centre for Cell Biology is supported by core grant 203149 from Wellcome . This work was supported by the Edinburgh Protein Production Facility (EPPF) and the Centre Core Grants ( 092076 and 203149 ) to the Wellcome Centre for Cell Biology at the University of Edinburgh . Publisher Copyright: {\textcopyright} 2022 The Author(s)",

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doi = "10.1016/j.xpro.2022.101490",

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AU - Pantier, Raphaël

AU - Chhatbar, Kashyap

AU - Alston, Grace

AU - Lee, Heng Yang

AU - Bird, Adrian

N1 - Funding Information: We thank Arttu Jolma and Jussi Taipale for useful advice regarding HT-SELEX library design. We thank Vladimir Benes and the EMBL Genecore Facility for technical support with high-throughput sequencing and library design. This work was funded by European Research Council Advanced Grant EC 694295 Gen-Epix and Wellcome Investigator Award 107930 to A.B., who is also a member of the Simons Initiative for the Developing Brain. The Centre for Cell Biology is supported by core grant 203149 from Wellcome . This work was supported by the Edinburgh Protein Production Facility (EPPF) and the Centre Core Grants ( 092076 and 203149 ) to the Wellcome Centre for Cell Biology at the University of Edinburgh . Publisher Copyright: © 2022 The Author(s)

PY - 2022/9/16

Y1 - 2022/9/16

N2 - 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).

AB - 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).

KW - bioinformatics

KW - high throughput screening

KW - molecular biology

KW - sequence analysis

KW - sequencing

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ER -

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

Abstract

Keywords / Materials (for Non-textual outputs)

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Projects

Core funding for the Wellcome Centre for Cell Biology

Wellcome Centre for Cell Biology

Towards understanding and treatment of MeCP2-related disorders

Cite this