Project description:Epigenetic mechanisms such as histone modifications and DNA methylation form a critical layer of control that regulates gene activities. Histone modifications play critical roles in adaptive tuning of chromatin structures. Profiling various histone modifications at the genome scale using primary tissues from animal and human samples is an important step for functional studies of epigenomes and epigenomics-based precision medicine. Because the profile of a histone mark is highly specific to a particular cell type, isolation of a cell type of interest from primary tissues is often necessary to generate a homogeneous cell population and such operation tends to yield a low number of cells. In addition, high-throughput processing is desired in such effort due to the number of histone marks of interests and the potential volume of samples in a hospital setting. In this protocol, we describe detailed information on device fabrication, setup, and operation of microfluidic oscillatory washing-based chromatin immunoprecipitation followed by sequencing (MOWChIP-seq) for profiling histone modifications using as few as 30-100 cells per assay with a throughput as high as 8 assays in a run. The critical step of MOWChIP-seq operation involves flowing of chromatin fragments through a packed bed of antibody-coated beads followed by a vigorous microfluidic oscillatory washing. The ChIP process is semi-automated for reduced labor and improved reproducibility. We have used the protocol to study a number of histone modifications in various types of mouse and human tissue types ranging from isolated nuclei of brain cells to cell subtypes isolated from human breast tissues.
Project description:Drug development is plagued by inefficiency and high costs due to issues such as inadequate drug efficacy and unexpected toxicity. Mass spectrometry (MS)-based proteomics, particularly isobaric quantitative proteomics, offers a solution to unveil resistance mechanisms and unforeseen side effects related to off-targeting pathways. Thermal proteome profiling (TPP) has gained popularity for drug target identification at the proteome scale. However, it involves experiments with multiple temperature points, resulting in numerous samples and considerable variability in large-scale TPP analysis. We propose a high-throughput drug target discovery workflow that integrates single-temperature TPP, a fully automated proteomics sample preparation platform (autoSISPROT), and Data Independent Acquisition (DIA) quantification. The autoSISPROT platform enables the simultaneous processing of 96 samples in less than 2.5 hours, achieving protein digestion, desalting, and optional TMT labeling (requires an additional 1 hour) with 96-channel all-in-tip operations. The results demonstrated excellent sample preparation performance with >94% digestion efficiency, >98% TMT labeling efficiency, and >0.9 of intraand inter-batch Pearson correlation coefficients. By automatically processing 87 samples, we identified both known targets and potential off-targets of 20 kinase inhibitors, affording over a 10-fold improvement in throughput compared to classical TPP. This fully automated workflow offers a high-throughput solution for proteomics sample preparation and drug target/off-target identification.
Project description:Queuosine (Q) is a conserved tRNA modification at the wobble anticodon position of tRNAs that read the codons of amino acids Tyr, His, Asn, and Asp. Q-modification in tRNA plays important roles in the regulation of translation efficiency and fidelity. Queuosine tRNA modification is synthesized de novo in bacteria, whereas the substrate for Q-modification in tRNA in mammals is queuine, the catabolic product of the Q-base of gut bacteria. This gut microbiome dependent tRNA modification may play pivotal roles in translational regulation in different cellular contexts, but extensive studies of Q-modification biology are hindered by the lack of high throughput sequencing methods for its detection and quantitation. Here, we describe a periodate-treatment method of biological RNA samples that enables single base resolution profiling of Q-modification in tRNAs by Nextgen sequencing. Periodate oxidizes the Q-base, which results in specific deletion signatures in the RNA-seq data. Unexpectedly, we found that periodate-treatment also enables the detection of several 2-thio-modifications including τm5s2U, mcm5s2U, cmnm5s2U, and s2C by sequencing in human and E. coli tRNA. We term this method Periodate-dependent analysis of queuosine and thio modification sequencing (PAQS-seq). We assess Q- and 2-thio-modifications at the tRNA isodecoder level, and 2-thio modification changes in stress response. PAQS-seq should be widely applicable in the biological studies of Q- and 2-thio-modifications in mammalian and microbial tRNAs.