"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. Building on ChIP-Rx benchmarking of creating a titration of epitope levels, we carry out a similar titration experiment of histone acetylation. Mitotically-arrested cells (low H3K9ac) were mixed with unsynchronized cells (high H3K9ac) in six ratios (0/100, 5/95, 25/75, 50/50, 75/25, 100/0). To all samples, a constant amount of two spike-in species chromatin (D. melanogaster and S. cerevisiae) were added. All replicates are technical replicates, deriving from the same pool of input chromatin. We next examine the operation limits of the approach over a large variation in the ratio of spike-in and target chromatin. We measure mitotic ChIP-seq H3K9ac signal in TSA or DMSO treated cells, varying spike-in amount from 1-10000x. Mitotically arrested cells were treated with either TSA (increasing acetylation) or DMSO control. For each set of treatments, spike-in was varied over five orders of magnitude (0.00025 to 2.5x spike-in/target ratio). Input samples ("input") contain the sheared chromatin that was not used for ChIP and serve as a control to identify non-specific signal.