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A low-input microfluidic ChIRP-seq technology for studying endogenous lncRNA binding

用于研究内源性 lncRNA 结合的低输入微流控 ChIRP-seq 技术

基本信息

批准号：
10400160
负责人：
Chang Lu
金额：
$ 26.31万
依托单位：
VIRGINIA POLYTECHNIC INST AND ST UNIV
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-05-01 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10400160
关键词：
Antisense DNA Binding Binding Sites Biological Assay Biological Process Biology Brain Neoplasms Cell Line Cell Nucleus Cells Chromatin Clinical Code Complex Cues DNA Development Disease Future Gene Expression Genome Genomic DNA Length Location Lung MALAT1 gene Manuals Methods Microfluidic Microchips Microfluidics Molecular Morphologic artifacts Mus Oligonucleotide Probes Patients Play Process Proteins Protocols documentation RNA Binding RNA purification Regulation Reproducibility Role Sampling Signal Transduction Stimulus System Technology Testing Tissue Sample Tissues Untranslated RNA Ursidae Family Work cell type chromatin isolation by RNA purification sequencing crosslink epigenomics genome-wide genome-wide analysis high throughput technology individual patient magnetic beads microfluidic technology operation overexpression parallel processing precision medicine scaffold tool tumor

项目摘要

Project Summary Long non-coding RNAs (lncRNAs) are >200 bp in length and bear no protein-coding potential. About 27,000 lncRNAs have been identified so far, most of which have versatile or unknown biological functions. LncRNAs have cell-type-specific expression that responds to environmental stimuli and developmental cues. lncRNAs may serve as molecular signals, decoys, guides, and scaffolds during their regulatory processes. Some lncRNAs are known to play important roles in development and diseases. Thus there is clear potential that they participate widely in the regulation of chromatin states and gene expression. Mapping of lncRNA binding sites in the genome is particularly important for understanding their regulatory roles. Over the years, a number of methods were developed to study genome-wide lncRNA-chromatin binding. ChIRP-seq (Chromatin Isolation by RNA purification) uses antisense DNA oligonucleotide probes to capture crosslinked and fragmented chromatin-lncRNA complexes before the purified genomic DNA is sequenced for lncRNA binding locations. Although ChIRP-seq has been gaining popularity, the method is plagued by several major issues including requirements of a huge number of starting cells, lncRNA overexpression, and tedious manual operation. In this project, we will develop a low-input version of ChIRP-seq that allows testing using 10K-100K starting cells, compared to tens of millions of cells required by current ChIRP-seq assay. This microfluidic ChIRP-seq technology will pave the way for studies in primary cells and tissues with endogenous lncRNA level and native lncRNA-chromatin interactions that bear direct biomedical relevance. Furthermore, the dramatic decrease in the required input will also allow cell-type-specific profiling of lncRNA binding which is critical for understanding cell-type-specific regulatory mechanisms. Our microfluidic technology offers much more reproducible, precise and effective manipulation of magnetic beads during pulldown of lncRNA-chromatin complexes than manual operation. The platform also offers fully automated and high-throughput processing, which will be important for the eventual processing of a large number of patient samples in clinical setting.

项目总结