Project description:By combining chromatin immunoprecipitation (ChIP) with an exonuclease that digests the ends of protein-bound DNA fragments, ChIP-exo characterizes genome-wide protein-DNA interactions at near base-pair resolution. However, the widespread adoption of ChIP-exo has been hindered by several technical challenges, including lengthy protocols, the need for multiple custom reactions, and incompatibilities with recent Illumina sequencing platforms. To address these barriers, we systematically optimized and adapted the ChIP-exo library construction protocol for the unique requirements of mammalian cells and current sequencing technologies. We introduce a Mammalian-Optimized ChIP-exo (MO-ChIP-exo) protocol that builds upon previous ChIP-exo protocols with systematic optimization of crosslinking, harvesting, and library construction. We validate MO-ChIP-exo by comparing it to previously published ChIP-exo protocols and demonstrate its adaptability to both suspension (K562) and adherent (HepG2, mESC) cell lines. This improved protocol provides a more robust and efficient method for generating high-quality ChIP-exo libraries from mammalian cells.
Project description:We propose an optimized protocol to reconstruct bacterial transcriptional regulatory networks (TRNs) using ChIP-exo and RNA-seq datasets. For the reconstruction of TRNs, the omics datasets were targeted to RpoS and generated in Escherichia coli K-12 MG1655 cultured at mid-exponential phase in M9 glucose media.
Project description:We propose an optimized protocol to reconstruct bacterial transcriptional regulatory networks (TRNs) using ChIP-exo and RNA-seq datasets. For the reconstruction of TRNs, the omics datasets were targeted to RpoS and generated in Escherichia coli K-12 MG1655 cultured at mid-exponential phase in M9 glucose media.
Project description:An Optimized Methods for Reconstruction of Transcriptional Regulatory Networks in Bacteria Using ChIP-exo and RNA-seq Datasets [ChIP-exo]
Project description:The emerging evidences support that exosome cargo miRNAs function as important regulators in cell differentiation. Therefore, in order to figure out the mechanism that Exo-AT mediated adipogenesis, we profiled miRNAs in Exo-AT using high-throughput sequencing (miRNA-seq). After trimming low-quality reads, contaminants, adaptors, and reads smaller than 15 nt, the remaining reads were mapped to merged pre-miRNA data bases. To identify the conserved miRNAs in Exo exosomes, miRNAs were aligned to miRBase v21. 148 and 154 types of known miRNAs in Exo-ADSCs and Exo-AT, respectively, were identified in the two replicates. Among these miRNAs, 103 miRNAs were simultaneously detected in both Exo-ADSCs and Exo-AT. Compared to Exo-ADSCs, 45 conserved miRNAs were enriched (expressed ≥ 2 folds, FDR<0.05) in Exo-AT. KEGG Pathway analysis was performed for the targets of the most 20 enriched miRNAs in Exo-AT (compared with Exo-ADSCs) to determine their potential function. Data showed that pathways that regulate adipogenesis such as Wnt signaling pathway, Insulin signaling pathway, MAPK signaling pathway, TGF-ß signaling pathway were enriched significantly for targets of Exo-AT miRNAs. Furthermore, 14 of 45 enriched miRNAs in Exo-AT (31.11%, such as miR-30a-5p, miR-148a-3p) were reported to participate in regulation of adipogenesis while 8 miRNAs (17.78%, such as miR-93-5p, miR-150-3p) that negatively control osteoblastic differentiation of MSC have been described.
Project description:Loss of contractility and acquisition of an epithelial phenotype of vascular smooth muscle cells (VSMCs) are key events in proliferative vascular pathologies such as atherosclerosis and post-angioplastic restenosis. There is no proper cell culture system allowing VSMC differentiation so that it is difficult to delineate the molecular mechanism responsible for proliferative vasculopathy. We investigated whether a micro-patterned substrate could restore the contractile phenotype of VSMCs in vitro. To induce and maintain the differentiated VSMC phenotype in vitro, we introduced a micro-patterned groove substrate to modulate the morphology and function of VSMCs.
Project description:Macrophage-derived exosomes hold significant promise for clinical disease therapy. Macrophages can be categorized into several subtypes. The resting state is known as the M0 subtype, and the exosomes they secrete are termed M0-Exo. Macrophages can be polarized to the M2 subtype by combined induction with IL-4 and IL-13, and the exosomes derived from M2 macrophages are referred to as M2-Exo. miRNAs are important cargo within exosomes. Since both M0-Exo and M2-Exo exhibit therapeutic effects in inflammatory responses, we performed miRNA sequencing on the contents of M0-Exo and M2-Exo to investigate the underlying mechanisms of their therapeutic actions.