Microfluidic low-input fluidized-bed enabled ChIP-seq device for automated and parallel analysis of histone modifications

Genome-wide epigenetic changes such as histone modifications form a critical layer of gene regulations and have been implicated in a number of different disorders such as cancer and inflammation. Progress has made to decrease the input required for gold-standard genome-wide profiling tools like chromatin immunoprecipitation followed by next generation sequencing (i.e. ChIP-seq) to allow scarce primary tissues of specific type from patients and lab animals to be tested. However, there has been very little effort to rapidly increase the throughput of these low-input tools. In this report, we demonstrate LIFE-ChIP-seq (Low-Input Fluidized-bed Enabled Chromatin Immunoprecipitation combined with sequencing), an automated and high-throughput microfluidic platform capable of running multiple sets of ChIP assays in as little as 1 h with as few as 50 cells per assay. Our technology will enable testing of a large number of samples and replicates with low-abundance primary samples in the context of precision medicine. Overall design: We examined genome-wide H3K4me3 profiles of GM12878 cell line starting with 1000 cells down to as few as 50 cells per assay. The ChIP experiment was performed using an automated microfluidic device (LIFE-ChIP), which performed 4 replicate experiments in every run under the same operating conditions. We also demonstrated that the device was capable of profiling two histone marks (H3K4me3 and H3K27ac) in one run with two replicates on each mark. The file name contains information on "cell number per assay"_"loading volume in microliters"_"washing time in minutes"_"no. of chamber"_"no. of device/histone mark (in the case of two marks being tested)". For example, "300_120L_20W_C1_H3K4me3" refers to the dataset taken by having 300 cells per assay, using 120 microliters of loading volume, 20 min of washing per buffer, and chamber 1 for characterizing H3K4me3 among the two histone marks profiled in the same run.全基因组表观遗传变化（如组蛋白修饰）是基因调控的关键层级，与癌症、炎症等多种疾病的发生发展密切相关。目前，针对染色质免疫沉淀结合下一代测序（chromatin immunoprecipitation followed by next generation sequencing, ChIP-seq）这类金标准全基因组谱分析工具，其所需的起始输入量已有所降低，使得研究者能够对患者及实验动物的稀有特定原代组织开展检测。然而，针对这类低输入量工具的通量提升研究却极为匮乏。本研究报道了LIFE-ChIP-seq（低输入流化床型染色质免疫沉淀联合测序技术，Low-Input Fluidized-bed Enabled Chromatin Immunoprecipitation combined with sequencing），这是一种自动化高通量微流控平台，可在1小时内完成多组ChIP实验，每组实验仅需低至50个细胞。该技术可实现精准医学背景下，对低丰度原代样本进行多样本、重复实验检测。 总体实验设计：我们针对GM12878细胞系的全基因组H3K4me3图谱展开检测，每组实验的起始细胞数从1000个低至50个。ChIP实验采用自动化微流控设备（LIFE-ChIP）完成，每次运行可在相同操作条件下完成4次重复实验。我们还证实，该设备可在单次运行中同时分析两种组蛋白修饰（H3K4me3与H3K27ac），每种修饰各完成2次重复实验。 文件名编码规则：文件名包含如下信息："每组实验细胞数_上样体积（微升）_洗涤时间（分钟）_腔室编号_实验标记/组蛋白修饰类型（若同时检测两种修饰则标注）"。例如，"300_120L_20W_C1_H3K4me3"代表该数据集的实验参数为：每组实验含300个细胞，上样体积为120微升，每步洗涤耗时20分钟，使用腔室1检测本次运行中同时分析的两种组蛋白修饰之一的H3K4me3。