Project description:We performed an unsupervised, high-throughput (HT) sequencing-cell SELEX (systematic evolution of ligands by exponential enrichment) using the MDSC-derived cell line MSC2. Specifically untreated MSC2 and IL4 treated MSC2 were used as negative and positive selector respectively. Selection was started from a 5 ug of 40bp variable region linked to two constant region.
Project description:total RNA from mouse (male c57BL/6) spleen labeled with Cy3 vs total RNA from mouse (male c57BL/6) B cells treated with IL4 labeled with Cy5- time course with repeats Keywords: ordered
Project description:Cancer biomarker discovery constitutes a frontier in cancer research. In recent years, cell-binding aptamers have become useful molecular probes for biomarker discovery. However, there are few successful examples, and the critical barrier resides in the identification of the cell-surface protein targets for the aptamers, where only a limited number of aptamer targets have been identified so far. Herein, we developed a universal SILAC-based quantitative proteomic method for target discovery of cell-binding aptamers. The method allowed for distinguishing specific aptamer-binding proteins from non-specific proteins based on abundance ratios of proteins bound to aptamer-carrying bait and control bait. In addition, we employed fluorescently labeled aptamers for monitoring and optimizing the binding conditions. We were able to identify and validate selectin L and integrin 4 as the protein targets for two previously reported aptamers, Sgc-3b and Sgc-4e, respectively. This strategy should be generally applicable for the discovery of protein targets for other cell-binding aptamers, which will promote the applications of these aptamers.
Project description:Purpose: Identify genes regulated by ALOX15 in A549 cells that were treated with +/- IL4 and +/- ALOX15 siRNA by Next-gen sequencing
Project description:Temporal analysis (60, 180, 360 min) of B cells treated with CD40 alone, IL4 alone or CD40 and IL4 (all in triplicates). Keywords: other
Project description:Purpose: Identify genes regulated by ALOX15 in Normal human bronchial epithelial (NHBE) cells that were treated with +/- IL4 and +/- ALOX15 siRNA by Next-gen sequencing
Project description:Here, we report an ssDNA aptamer with high specificity and affinity towards Salmonella paratyphi A generated using the whole-cell SELEX process. The aptamers generated against an organism show salient features, such as higher affinity than existing antibodies, and are highly specific towards the targeted organism. Thus, the generated aptamer sequences can serve as potential biomarkers for the onsite detection of pathogens with high specificity and sensitivity. Molecular dynamics simulation was used to model the linear chain of the aptamers to a three-dimensional conformation, and the binding mechanism against DNA gyrase was established.
Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived IL4/IL13 treated BMDMs transcriptome profiling (RNA-seq) to quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods. Methods: BMDMs mRNA profiles of 8 weeks old wild-type (WT) mice were generated, stimulated with IL4/IL13, IL4/IL13+ TNF or TNF, deep sequenced, in triplicate, using Illumina NovaSeq 6000 platform. Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm9). Conclusions: Our study represents the first detailed analysis of IL4/IL13 or IL4/IL13+TNF stimulated BMDMs, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.
Project description:The ability to detect and target β cells in vivo can drastically refine the way diabetes is studied and treated. By an unsupervised Systematic evolution of ligands by exponential enrichment (SELEX) we identified two RNA aptamers that specifically recognize mouse and human β cells in vitro and in vivo. Here we took advantage of commercially available high density protein arrays to identify putative target of the two islet specific aptamers. Briefly, 5' biotynilated RNA aptamer 1-717 and m12-3773 were chemically produced , complexed with Alexafluor 647-streptavidin and used as probe on the HuProt™ v2.0 19K protein array. Putative binders were further confirmed by cold target inhibition assays, silencing experiments, and surface plasmon resonance.