Multiplexed chromatin immunoprecipitation sequencing for quantitative study of histone modifications and chromatin factors

被引:0
|
作者
Kumar, Banushree [1 ,2 ,3 ]
Navarro, Carmen [1 ,2 ,3 ]
Yung, Philip Yuk Kwong [1 ,2 ]
Lyu, Jing [1 ,2 ]
Mantero, Angelo Salazar [1 ,2 ]
Katsori, Anna-Maria [1 ,2 ]
Schwaemmle, Hannah [1 ,2 ]
Martin, Marcel [4 ]
Elsaesser, Simon J. [1 ,2 ]
机构
[1] Karolinska Inst, Dept Med Biochem & Biophys, Sci Life Lab, Solna, Sweden
[2] Karolinska Inst, Stockholm Node, Ming Wai Lau Ctr Reparat Med, Solna, Sweden
[3] Working Lab, Epigen AB, Solna, Sweden
[4] Stockholm Univ, Dept Biochem & Biophys, Sci Life Lab, Natl Bioinformat Infrastruct Sweden, Solna, Sweden
基金
瑞典研究理事会;
关键词
CHIP-SEQ; STATE;
D O I
10.1038/s41596-024-01058-z
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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. MINUTE-ChIP is a sensitive multiplexed ChIP method that quantitatively profiles relative differences in genome-wide epigenetic patterns across multiple samples and conditions. In a MINUTE-ChIP workflow, samples are ligated in a one-pot reaction to UMI-containing adapters before pooling and splitting into multiple parallel immunoprecipitation reactions.The multiplexed MINUTE-ChIP sequencing files can be readily analyzed by using a comprehensive analysis pipeline that generates output files for direct, quantitative comparison across conditions. MINUTE-ChIP is a multiplexed chromatin immunoprecipitation and sequencing method that measures global and locus-specific changes in histone modification patterns and chromatin factor binding across multiple samples and conditions.
引用
收藏
页码:779 / 809
页数:31
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