Protocol for using heterologous spike-ins to normalize for technical variation in chromatin immunoprecipitation

Franziska Greulich; Aikaterini Mechtidou; Teresa Horn; Nina Henriette Uhlenhaut

doi:10.1016/j.xpro.2021.100609

Protocol for using heterologous spike-ins to normalize for technical variation in chromatin immunoprecipitation

STAR Protoc. 2021 Jun 16;2(3):100609. doi: 10.1016/j.xpro.2021.100609. eCollection 2021 Sep 17.

Authors

Franziska Greulich¹, Aikaterini Mechtidou², Teresa Horn², Nina Henriette Uhlenhaut^{1

2}

Affiliations

¹ Metabolic Programming, School of Life Sciences Weihenstephan, ZIEL-Institute for Food & Health, Technische Universitaet Muenchen (TUM), 85354 Freising, Germany.
² Institute for Diabetes and Obesity (IDO) & Institute for Diabetes and Cancer (IDC), Helmholtz Center Munich (HMGU) and German Center for Diabetes Research (DZD), 85764 Neuherberg (Munich), Germany.

Abstract

Quantifying differential genome occupancy by chromatin immunoprecipitation (ChIP) remains challenging due to variation in chromatin fragmentation, immunoprecipitation efficiencies, and intertube variability. In this protocol, we add heterologous spike-ins from Drosophila chromatin as an internal control to the mice chromatin before immunoprecipitation to normalize for technical variation in ChIP-qPCR or ChIP-seq. The choice of spike-in depends on the evolutionary conservation of the protein of interest and the antibody used. For complete details on the use and execution of this protocol, please refer to Greulich et al. (2021).

Keywords: Cell Biology; ChIPseq; Chromatin immunoprecipitation (ChIP); Molecular Biology; Sequence analysis.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Binding Sites
Chromatin Immunoprecipitation / methods*
Drosophila melanogaster
High-Throughput Nucleotide Sequencing / methods
Mice
Proteins / metabolism*
Real-Time Polymerase Chain Reaction

Substances

Proteins