PIXUL-ChIP: integrated high-throughput sample preparation and analytical platform for epigenetic studies

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.

Focused ultrasound extraction (FUSE) for the rapid extraction of DNA from tissue matrices

“…This mechanism is similar to how sonic cleaners have been used to break up bacteria for DNA analysis (Taylor et al., 2001). In addition, other focused ultrasonicators have also recently been used for library preparation (Bomsztyk et al., 2019) and sample preparation (Kresse et al., 2018), including testing of clinical sample types and cancer genomics. The histotripsy transducers investigated here offer the potential for further advancing ultrasonic DNA extraction by delivering much higher peak negative pressures and creating dense bubble clouds capable of breaking down even very difficult tissue types.…”

Computer Designed PRC2 Inhibitor, EBdCas9, Reveals Functional TATA boxes in Distal Promoter Regions

“…To dissect the mechanism of EBdCas9 action at precise genomic locus, we used ChIP-qPCR (PIXUL-Matrix-ChIP 47 ) to analyze the epigenetic landscape of TBX18 g6 targeted region. As above, EBdCas9 or NCdCas9 expression was induced in iPSC (WTC) using doxycycline followed by TBX18 gRNA6 (g6) transfections at day 0 and day 1 and analysis on day 3 by RT-qPCR and ChIP-qPCR ( Fig 2A ).…”

“…Matrix-ChIP was performed as previously described 47, 84 . Briefly, UV treated 96-well polypropylene microplates were incubated with protein A on a low-speed shaker at room temperature overnight.…”

“…For each qPCR run the primer efficiency curve was fit to cycle threshold (CT) versus log (genomic DNA concentration) using an r-squared best fit. DNA concentration values for each ChIP and input chromatin DNA sample were calculated from their respective average Ct values as previously described 47, 84 . Final results are expressed as fraction of input DNA versus the normalization antibody H3 relative to no guide (-g).…”

Genome-Bound Enzymes as Epigenetic Drug Targets in Cancer

“…Thus, the ability to accurately assess epigenetic profiles and chromatin-bound modifiers is critically important for designing new cancer treatments. Although the list of chromatin modifying enzymes is extensive, advances in ChIP assays have overcome the challenges to study low abundance gene-bound proteins allowing high throughput parallel detection of dozens enzymes at loci of interest [8,9]. Many drugs, including selective agents, have been introduced that target DNA and histone modifying enzymes including EZH2 (H3K27me methyltransferase), DOT1L (H3K79me methyltransferase), HDACs (histone deacetylases), DNMTs (DNA methyltransferases) and ERKs (protein kinases).…”