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

项目摘要 长的非编码RNA(LncRNAs)长度为200个碱基对，不具有蛋白质编码潜力。约27,000人 到目前为止，已发现的lncRNAs大多具有多种或未知的生物学功能。IncRNAs 具有对环境刺激和发育线索作出反应的特定细胞类型的表达。IncRNAs 在它们的调控过程中，可以作为分子信号、诱饵、引导和支架。一些人 众所周知，lncRNAs在发育和疾病中发挥重要作用。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 对于理解它们在基因组中的调节作用尤为重要。多年来，一些 建立了研究全基因组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化 在纯化的基因组DNA被测序之前，染色质-lncRNA复合体被用于测定lncRNA的结合位置。 尽管chirp-seq越来越受欢迎，但该方法受到几个主要问题的困扰，包括 要求起始细胞数量庞大，lncRNA过度表达，手工操作繁琐。在这 项目，我们将开发一个低输入版本的chirp-seq，允许使用10K-100K起始电池进行测试， 与目前的chirp-seq分析所需的数千万个细胞相比。这种微流控线性调频序列 技术将为在原代细胞和组织中进行内源性lncRNA水平和天然的研究铺平道路 具有直接生物医学相关性的lncRNA-染色质相互作用。此外，中国经济的急剧下降 所需的输入还将允许对特定细胞类型的lncRNA结合进行分析，这对于理解 特定细胞类型的调节机制。我们的微流控技术提供了更多的重复性、精确度 在下拉lncRNA-染色质复合体过程中对磁珠的有效操作比手动操作更有效 手术。该平台还提供完全自动化和高吞吐量的处理，这对 在临床环境中对大量患者样本的最终处理。

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.