Probing cell identity hierarchies by fate titration and collision during direct reprogramming [scRNA-seq & scATAC-seq]

Collide-seq: A systematic comparison of how different reprogramming transcription factors achieve conversion as well as an in-depth analysis of how cells resolve the conflict when more than one fate is instructed. Mouse embryonic fibroblasts were nucleofected with doxycycline inducible constructs for Ascl1, MyoD1 and a DNA binding mutant of Ascl1 (mutAscl1). Cells were expanded for about 10 days and positive cells sorted using FACS. All conditions were pooled and plated together. Transcription factor expression was induced for 72h via the administration of doxycycline. After 72h, cells were collected for scMultiome, i.e., scRNA-seq and scATAC-seq from the same cells

Collide-seq：一项系统性比较不同重编程转录因子如何介导细胞命运转化，并深入解析细胞在接收到多重命运指令时如何解决冲突的数据集。本研究以小鼠胚胎成纤维细胞为材料，通过核转染技术导入分别搭载Ascl1、MyoD1以及Ascl1的DNA结合突变体（mutAscl1）的多西环素诱导型表达载体。将转染后的细胞扩增约10天，随后通过荧光激活细胞分选术（Fluorescence-Activated Cell Sorting，FACS）筛选阳性细胞。将所有实验组的细胞混合后统一接种培养，通过添加多西环素诱导转录因子表达，诱导时长为72小时。诱导72小时后，收集细胞进行单细胞多组学测序（scMultiome），即对同一批细胞同时开展单细胞RNA测序（scRNA-seq）与单细胞转座酶可及性测序（scATAC-seq）。