Protocol for integrative analysis of transcription factor-nucleosome interactions using SeEN-seq and cryo-EM structure determination

TL;DR

This paper introduces SeEN-seq, a protocol to identify where transcription factors bind on nucleosomes, aiding in understanding gene regulation.

Contribution

The novel contribution is the SeEN-seq protocol for mapping TF-binding sites on nucleosomes and preparing samples for cryo-EM.

Findings

SeEN-seq enables systematic identification of TF-binding sites on nucleosomes.

The protocol allows preparation of homogeneous TF-nucleosome complexes for cryo-EM analysis.

Abstract

Pioneer transcription factors (TFs) possess the ability to read out DNA motifs embedded within nucleosomes, driving changes in gene expression during cellular differentiation and reprogramming. Here, we present selected engagement on nucleosome sequencing (SeEN-seq), a protocol designed to systematically identify potential TF-binding sites on the nucleosome. We describe steps for nucleosome library assembly, SeEN-seq assay, and cryoelectron microscopy (cryo-EM) sample preparation. This protocol facilitates the preparation of homogeneous pioneer TF-nucleosome complexes for cryo-EM structure determination using single-particle analysis.

For complete details on the use and execution of this protocol, please refer to Michael et al.1

•Instructions for the preparation of an SeEN-seq DNA library by EcoRV digestion•Steps for purification using either the anion exchange column or Prepcell apparatus•Procedures for SeEN-seq data analysis•Guidance of TF-nucleosome complex preparation for cryo-EM analysis

Instructions for the preparation of an SeEN-seq DNA library by EcoRV digestion

Steps for purification using either the anion exchange column or Prepcell apparatus

Procedures for SeEN-seq data analysis

Guidance of TF-nucleosome complex preparation for cryo-EM analysis

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Pioneer transcription factors (TFs) possess the ability to read out DNA motifs embedded within nucleosomes, driving changes in gene expression during cellular differentiation and reprogramming. Here, we present selected engagement on nucleosome sequencing (SeEN-seq), a protocol designed to systematically identify potential TF-binding sites on the nucleosome. We describe steps for nucleosome library assembly, SeEN-seq assay, and cryoelectron microscopy (cryo-EM) sample preparation. This protocol facilitates the preparation of homogeneous pioneer TF-nucleosome complexes for cryo-EM structure determination using single-particle analysis.

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