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：
10594496
负责人：
Chang Lu
金额：
$ 26.27万
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/10594496
关键词：
Antisense DNA Binding Binding Sites Biological Assay Biological Process Biology Brain 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 Maps 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 Technology Assessment Testing Tissue Sample Tissues Untranslated RNA 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.

项目摘要长的非编码RNA（LNCRNA）的长度> 200 bp，并且没有蛋白质编码潜力。大约27,000 到目前为止，已鉴定出LNCRNA，其中大多数具有多功能或未知的生物学功能。 lncrnas 具有对环境刺激和发育提示的反应的细胞型特异性表达。 lncrnas 可以在调节过程中充当分子信号，诱饵，指南和支架。一些众所周知，LNCRNA在发育和疾病中起着重要作用。因此，他们有明确的潜力广泛参与染色质状态和基因表达的调节。 lncRNA结合位点的映射在基因组中，对于理解其调节作用尤为重要。多年来，许多开发了研究全基因组LNCRNA-染色质结合的方法。 chirp-seq（染色质分离通过RNA纯化）使用反义DNA寡核苷酸探针捕获交联和碎片在纯化的基因组DNA之前，将染色质 - lncRNA复合物测序以进行lncRNA结合位置。尽管Chirp-Seq一直在越来越受欢迎，但该方法受到了几个主要问题的困扰大量启动细胞，LNCRNA过表达和乏味的手动操作的要求。在这个项目，我们将开发一个低输入版本的Chirp-seq，该版本允许使用10K-100K启动单元格进行测试，与当前Chirp-Seq分析所需的数千万细胞相比。这个微流体chirp-seq 技术将为具有内源性lncRNA水平和天然的原代细胞和组织研究铺平道路具有直接生物医学相关性的LNCRNA-染色质相互作用。此外，急剧下降所需的输入还将允许LNCRNA结合的细胞类型特异性分析，这对于理解至关重要细胞型特异性调节机制。我们的微流体技术提供了更可重复，精确的与手动相比，在LNCRNA-染色质复合物下拉期间对磁珠的有效操纵手术。该平台还提供完全自动化和高通量处理，这对于在临床环境中最终处理大量患者样本。