"The Wild West of Spike-in Normalization": Benchmarking the ChIP-seq spike-in normalization method

Spike-in normalization is a powerful approach to assess global changes in data obtained from genomic mapping of DNA-associated proteins by methods such as ChIP-sequencing (ChIP-seq) or CUT&RUN. While multiple spike-in methods provide detailed documentation, the implementation of these approaches often omit critical quality control steps and veer from the established procedures. Here, we show that proper application of spike-in normalization can increase quantification accuracy across a spectrum of conditions. Next, we outline how misuse of spike-in approaches can create erroneous biological interpretations and provide guidelines to minimize pitfalls when applying this approach to normalize data from protein-DNA interaction results.