Single-cell analysis of chromatin silencing programs in development and tumor progression. Single-cell analysis of chromatin silencing programs in development and tumor progression

Single-cell analysis has become a powerful approach for the molecular characterization of complex tissues. Methods for quantifying gene expression and chromatin accessibility2 of single cells are now well-established, but analysis of chromatin regions with specific histone modifications has been technically challenging. Here, we adapt the recently published CUT&Tag method3 to scalable single-cell platforms to profile chromatin landscapes in single cells (scCUT&Tag) from complex tissues. We focus on profiling Polycomb Group (PcG) silenced regions marked by H3K27 trimethylation (H3K27me3) in single cells as an orthogonal approach to chromatin accessibility for identifying cell states. We show that scCUT&Tag profiling of H3K27me3 distinguishes cell types in human blood and allows the generation of cell-type-specific PcG landscapes from heterogeneous tissues. Furthermore, we use scCUT&Tag to profile H3K27me3 in a brain tumor patient before and after treatment, identifying cell types in the tumor microenvironment and heterogeneity in PcG activity in the primary sample and after treatment. Overall design: We used Cleavage under targets and Tagmentation (Cut-and-Tag), a chromatin profiling strategy in which antibody-targeted controlled integration of DNA sequencing adapters produce libraries in nuclei. The resulting nuclei were isolated either by a microwell approach (Takara’s iCell8) or by a microfluidic approach (10x Genomic’s GEM) where unique barcodes are added, followed by paired-end sequencing.

单细胞分析已成为复杂组织分子表征的强有力研究手段。目前，定量单细胞基因表达与染色质可及性（chromatin accessibility）的方法已较为成熟，但针对带有特定组蛋白修饰的染色质区域开展分析仍存在技术挑战。本研究将近期发表的CUT&Tag方法3适配至可扩展的单细胞平台，用于解析复杂组织单细胞的染色质全景（scCUT&Tag）。本研究聚焦于单细胞中由三甲基化组蛋白H3赖氨酸27（H3K27me3）标记的多梳蛋白家族（Polycomb Group, PcG）沉默区域的分析，以此作为染色质可及性分析的正交方法，用于鉴定细胞状态。研究表明，针对H3K27me3的scCUT&Tag分析可区分人血液中的细胞类型，并能从异质性组织中获取细胞类型特异性的PcG染色质全景。此外，本研究利用scCUT&Tag分析了一名脑肿瘤患者治疗前后的H3K27me3状态，鉴定出肿瘤微环境中的细胞类型，以及原发样本与治疗后样本中PcG活性的异质性。整体实验设计：本研究采用靶向切割与标签化（Cleavage under targets and Tagmentation, CUT&Tag）技术，该染色质分析策略通过抗体靶向介导的可控整合DNA测序接头，在细胞核中完成文库构建。后续通过微孔法（Takara的iCell8平台）或微流控法（10x Genomic’s GEM平台）分离带有唯一条形码标记的细胞核，随后进行双端测序。