Microfluidic MeDIP-seq for low-input methylomic analysis of mammary tumorigenesis in mice
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PROVIDER: PRJNA430738 | ENA |
REPOSITORIES: ENA
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Project description:Studies of dynamic epigenomic changes during tumorigenesis using mice often require profiling epigenomes using a tiny quantity of tissue samples. Conventional epigenomic tests do not support such analysis due to the large amount of materials required by these assays. In this study, we developed an ultrasensitive microfluidics-based methylated DNA immunoprecipitation followed by next-generation sequencing (MeDIP-seq) technology for profiling methylomes using as little as 0.5 ng DNA (or ~100 cells) with 1.5 h on-chip process for immunoprecipitation. This technology enabled us to examine genome-wide DNA methylation in a C3(1)/SV40 T-antigen transgenic mouse model during different stages of mammary cancer development. Using our data, we identified differentially methylated regions and their associated genes in different periods of cancer development. Our results showed that unique methylomic features were presented in various tumor developmental stages.
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Project description:Epigenetic profiling by chromatin immunoprecipitation followed by sequencing (ChIP-seq) has become a powerful tool for genome-wide identification of regulatory elements, for defining transcriptional regulatory networks and for screening for biomarkers. However, the ChIP-seq protocol for low-input samples is laborious, time-consuming and suffers from experimental variation, resulting in poor reproducibility and low throughput. Although prototypic microfluidic ChIP-seq platforms have been developed, these are poorly transferable as they require sophisticated custom-made equipment and in-depth microfluidic and ChIP expertise, while lacking parallelisation. To enable standardized, automated ChIP-seq profiling of low-input samples, we constructed microfluidic PDMS-based plates capable of performing 24 sensitive ChIP reactions within 30 minutes hands-on time and 4.5 hours machine-running time. These disposable plates can be conveniently loaded into a widely available controller for pneumatics and thermocycling. In light of the Plug and Play (PnP) ChIP plates and workflow, we named our procedure PnP-ChIP-seq. We demonstrate high-quality ChIP-seq on hundreds to few thousands of cells for all six post-translational histone modifications that are included in the International Human Epigenome Consortium set of reference epigenomes. PnP-ChIP-seq robustly detects epigenetic differences on promoters and enhancers between naïve and more primed mouse embryonic stem cells (mESCs). Furthermore, we used our platform to generate epigenetic profiles of rare subpopulations of mESCs that resemble the 2-cell stage of embryonic development. PnP-ChIP-seq allows non-expert labs worldwide to conveniently run robust, standardized ChIP-seq, while its high-throughput, consistency and sensitivity paves the way towards large-scale profiling of precious sample types such as rare subpopulations of cells or biopsies.
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Project description:Microfluidic platform for next-generation sequencing library preparation with low-input samples
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Project description:Genome-wide epigenetic changes such as histone modifications form a critical layer of gene regulations and have been implicated in a number of different disorders such as cancer and inflammation. Progress has made to decrease the input required for gold-standard genome-wide profiling tools like chromatin immunoprecipitation followed by next generation sequencing (i.e. ChIP-seq) to allow scarce primary tissues of specific type from patients and lab animals to be tested. However, there has been very little effort to rapidly increase the throughput of these low-input tools. In this report, we demonstrate LIFE-ChIP-seq (Low-Input Fluidized-bed Enabled Chromatin Immunoprecipitation combined with sequencing), an automated and high-throughput microfluidic platform capable of running multiple sets of ChIP assays in as little as 1 h with as few as 50 cells per assay. Our technology will enable testing of a large number of samples and replicates with low-abundance primary samples in the context of precision medicine.
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Project description:There has been an extensive effort underway to profile epigenetic features at the genome-wide scale using primary ex vivo tissues. Cell-type specificity of epigenomes calls for enrichment to obtain a homogenous cell population from a small quantity of tissues. Thus technologies that permit both ultralow input and high throughput are desired for profiling an array of histone marks. Here we demonstrate a simple microfluidic technology, SurfaceChIP-seq, for profiling genome-wide histone modifications using as few as 30 cells per assay and with up to 8 assays running in parallel. We applied the technology to study epigenomic landscapes in neurons and glia in prefrontal cortex and cerebellum of mouse brain. The data revealed extensive epigenomic difference in the two regions on important functional elements such as promoters and enhancers.
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Project description:Microfluidic devices provide a low-input and efficient platform for single-cell RNA-seq (scRNA-Seq). Here we present microfluidic diffusion-based RNA-seq (MID-RNA-seq) for conducting scRNA-seq with a diffusion-based reagent swapping scheme. This device incorporates cell trapping, lysis, reverse transcription and PCR amplification all in one microfluidic chamber. MID-RNA-Seq provides high data quality that is comparable to existing scRNA-seq methods while implementing a simple device design that permits multiplexing. The robustness and scalability of MID-RNA-Seq device will be important for transcriptomic studies of scarce cell samples.
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