Multiplexed chromatin immunoprecipitation sequencing for quantitative study of histone modifications and chromatin factors.
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Kumar B, Navarro C, Yung PYK, Lyu J, Salazar Mantero A, Katsori AM, Schwämmle H, Martin M, Elsässer SJ
Nat Protoc - (-) - [2024-10-03; online 2024-10-03]
ChIP-seq is a widely used technique for studying histone post-translational modifications and DNA-binding proteins. DNA fragments associated with a specific protein or histone modification epitope are captured by using antibodies, sequenced and mapped to a reference genome. Albeit versatile and popular, performing many parallel ChIP-seq experiments to compare different conditions, replicates and epitopes is laborious, is prone to experimental variation and does not allow quantitative comparisons unless adequate spike-in chromatin is included. We present a detailed protocol for performing and analyzing a multiplexed quantitative chromatin immunoprecipitation-sequencing experiment (MINUTE-ChIP), in which multiple samples are profiled against multiple epitopes in a single workflow. Multiplexing not only dramatically increases the throughput of ChIP-seq experiments (e.g., profiling 12 samples against multiple histone modifications or DNA-binding proteins in a single experiment), but also enables accurate quantitative comparisons. The protocol consists of four parts: sample preparation (i.e., lysis, chromatin fragmentation and barcoding of native or formaldehyde-fixed material), pooling and splitting of the barcoded chromatin into parallel immunoprecipitation reactions, preparation of next-generation sequencing libraries from input and immunoprecipitated DNA and data analysis using our dedicated analysis pipeline. This pipeline autonomously generates quantitatively scaled ChIP-seq tracks for downstream analysis and visualization, alongside necessary quality control indicators. The entire workflow requires basic knowledge in molecular biology and bioinformatics and can be completed in 1 week. MINUTE-ChIP empowers biologists to perform every ChIP-seq experiment with an appropriate number of replicates and control conditions, delivering more statistically robust, exquisitely quantitative and biologically meaningful results.
PubMed 39363107
DOI 10.1038/s41596-024-01058-z
Crossref 10.1038/s41596-024-01058-z
pii: 10.1038/s41596-024-01058-z