Clonal multi-omics reveals Bcor as a regulator of dendritic cell development and its action at a clonal level. Clonal multi-omics reveals Bcor as a regulator of dendritic cell development and its action at a clonal level

Despite major developments in single cell multi-omics approaches, a single stem or progenitor cell can only be tested once. Here we develop ‘clonal multi-omics’, where recent daughters of a clone act as surrogates of the parental cell allowing multiple tests of its molecular profile and/or cellular fate under different conditions. We first present SIS-seq – a novel clonal method whereby siblings are examined in parallel for gene expression by RNA-seq, or for fate after culture. We use this method to identify, then validate using CRISPR, the genes expressed in single haematopoietic progenitors that control the development of the different dendritic cell (DC) subtypes. In this way, Bcor is identified as a suppressor of plasmacytoid DC (pDC) and conventional DC subtype 2 (cDC2) numbers during Flt3 ligand-mediated ‘emergency’ DC development. Using a complementary method SIS-skew – where WT and BcorKO siblings of the same clone are examined in parallel for fate – we discover that Bcor restricts clonal expansion, especially for generation of cDC2s, and suppresses clonal fate potential, especially for pDCs. SIS-seq and SIS-skew therefore represent novel and broadly applicable clonal multi-omics approaches that could reveal the molecular and cellular mechanisms governing stem, immune, reprogrammed and cancer cell function at a single cell level, including after genetic or extrinsic factor perturbation. Overall design: Mouse HSPCs were isolated from bone marrow and sorted into 384 PCR plate, with library generated using the CEL-seq2 protocol.

尽管单细胞多组学技术已取得重大进展，但单个干细胞或祖细胞仅能接受一次检测。在此，我们开发了‘克隆多组学’技术，即利用克隆的当代子代细胞作为亲代细胞的替代物，使其可在不同条件下接受多次分子特征与/或细胞命运检测。
我们首先推出SIS-seq——一种全新的克隆方法，可平行检测同一克隆的子代细胞的RNA-seq基因表达谱，或培养后的细胞命运。我们利用该方法先筛选出调控不同树突状细胞（dendritic cell, DC）亚型发育的、在单个造血祖细胞中表达的基因，随后通过CRISPR技术进行验证。通过此途径，我们发现Bcor是Flt3配体介导的‘应急’树突状细胞发育过程中，浆细胞样树突状细胞（plasmacytoid DC, pDC）与经典树突状细胞亚型2（conventional DC subtype 2, cDC2）数量的抑制因子。
我们还采用互补方法SIS-skew——平行检测同一克隆的野生型（Wild Type, WT）与Bcor敲除（BcorKO）子代细胞的细胞命运——进一步发现，Bcor可限制克隆扩增，尤其是在cDC2生成过程中，并抑制克隆的命运潜能，尤其是pDC的生成。
因此，SIS-seq与SIS-skew代表了全新且普适性广泛的克隆多组学技术，可用于揭示调控干细胞、免疫细胞、重编程细胞及癌细胞单细胞层面功能的分子与细胞机制，包括在遗传或外源性因子扰动后的机制。
整体实验设计：从小鼠骨髓中分离造血干细胞与祖细胞（hematopoietic stem and progenitor cells, HSPCs），并分选至384孔PCR板中，采用CEL-seq2流程构建测序文库。