Abstract
Chromatin immunoprecipitation (ChIP) is the most widely used approach for identification of genome-associated proteins and their modifications. We have previously introduced a microplate-based ChIP platform, Matrix ChIP, where the entire ChIP procedure is done on the same plate without sample transfers. Compared to conventional ChIP protocols, the Matrix ChIP assay is faster and has increased throughput. However, even with microplate ChIP assays, sample preparation and chromatin fragmentation (which is required to map genomic locations) remains a major bottleneck. We have developed a novel technology (termed ‘PIXUL’) utilizing an array of ultrasound transducers for simultaneous shearing of samples in standard 96-well microplates. We integrated PIXUL with Matrix ChIP (‘PIXUL-ChIP’), that allows for fast, reproducible, low-cost and high-throughput sample preparation and ChIP analysis of 96 samples (cell culture or tissues) in one day. Further, we demonstrated that chromatin prepared using PIXUL can be used in an existing ChIP-seq workflow. Thus, the high-throughput capacity of PIXUL-ChIP provides the means to carry out ChIP-qPCR or ChIP-seq experiments involving dozens of samples. Given the complexity of epigenetic processes, the use of PIXUL-ChIP will advance our understanding of these processes in health and disease, as well as facilitate screening of epigenetic drugs.
Highlights
The chromatin immunoprecipitation (ChIP) assay, a widely-used approach for identifying histone modifications and genome-associated proteins, is one of the most powerful tools to study transcription and epigenetics processes [1,2,3,4,5,6]
We have previously developed a highthroughput microplate ChIP assay, Matrix ChIP, which speeds up the analytical process, dramatically increases the assay’s throughput, and provides superior sensitivity and reproducibility as compared to other protocols [7,8,9]
To estimate ultrasound treatment efficiency without the confounding effect of DNA crosslinking, we first used purified salmon DNA, which is readily available in large quantities. 100 l of salmon DNA at 100 ng/l was aliquoted into each one of the 96 wells of two replicate plates
Summary
The chromatin immunoprecipitation (ChIP) assay, a widely-used approach for identifying histone modifications and genome-associated proteins, is one of the most powerful tools to study transcription and epigenetics processes [1,2,3,4,5,6]. Tissues samples from all these organs were resuspended in 100 l chromatin shearing buffer and added to wells of the 96-well plate for sonication in PIXUL using the same protocol as for cell culture.
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