Protocol for double-crosslinking ChIP-seq to improve data quality and enhance detection of challenging chromatin targets

Yu Bao; Roberta Cacioppo; Alastair Crisp; Ivan Shlamovitz; Julian E Sale; Ana Tufegdzic Vidakovic; Stephin J Vervoort; Andrew Zeller

doi:10.1016/j.xpro.2025.104171

Protocol for double-crosslinking ChIP-seq to improve data quality and enhance detection of challenging chromatin targets

STAR Protoc. 2025 Oct 29;6(4):104171. doi: 10.1016/j.xpro.2025.104171. Online ahead of print.

Authors

Yu Bao¹, Roberta Cacioppo¹, Alastair Crisp¹, Ivan Shlamovitz¹, Julian E Sale¹, Ana Tufegdzic Vidakovic², Stephin J Vervoort³, Andrew Zeller⁴

Affiliations

¹ MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, UK.
² MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, UK. Electronic address: atv@mrc-lmb.cam.ac.uk.
³ The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia.
⁴ MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, UK. Electronic address: azeller@mrc-lmb.cam.ac.uk.

Abstract

Chromatin factors drive genome function, yet many lack direct DNA-binding activity and cannot be conventionally profiled. Here, we present dxChIP-seq, a double-crosslinking chromatin immunoprecipitation sequencing (ChIP-seq) protocol that improves mapping of chromatin factors, including those that do not bind DNA directly, while enhancing signal-to-noise ratio. We describe steps for double-crosslinking, focused ultrasonication, immunoprecipitation, DNA purification, and library preparation. We then detail procedures for sequencing and analysis. This protocol is compatible with adherent cells and complex multicellular structures. For complete details on the use and execution of this protocol, please refer to Cacioppo et al.¹.

Keywords: ChIP; Chromatin immunoprecipitation; Genomics; Molecular Biology.