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

项目摘要 长非编码RNA（lncRNA）长度>200 bp，不具有蛋白质编码潜力。约2.7万 lncRNA已被鉴定出来，其中大部分具有多种或未知的生物学功能。lncRNAs 具有细胞类型特异性表达，对环境刺激和发育线索作出反应。lncRNAs 在其调控过程中可作为分子信号、诱饵、向导和支架。一些 已知lncRNA在发育和疾病中起重要作用。因此，他们显然有可能 广泛参与染色质状态和基因表达的调节。lncRNA结合位点的定位 对于理解它们的调节作用尤为重要。多年来， 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片段化的 在对纯化的基因组DNA测序以确定lncRNA结合位置之前，先测定染色质-lncRNA复合物。 虽然ChIRP-seq已经越来越受欢迎，但该方法受到几个主要问题的困扰，包括 需要大量的起始细胞、lncRNA过表达和繁琐的人工操作。在这 项目，我们将开发一个低输入版本的ChIRP-seq，允许使用10 K-100 K起始细胞进行测试， 相比之下，目前的ChIRP-seq检测需要数千万个细胞。这种微流体ChIRP-seq 这项技术将为在原代细胞和组织中进行内源性lncRNA水平和天然lncRNA水平的研究铺平道路。 具有直接生物医学相关性的lncRNA-染色质相互作用。此外， 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 细胞类型特异性调节机制。我们的微流控技术提供了更可重复的，精确的 并且在lncRNA-染色质复合物的下拉期间比手动更有效地操纵磁珠 操作该平台还提供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。

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.