10X Genomics single-cell RNA-Seq data set of CreER mice

The liver is organized into zones in which hepatocytes express different metabolic enzymes. The cells most responsible for liver repopulation and regeneration remain undefined because fate-mapping has only been performed on a few hepatocyte subsets. Fourteen mouse fate mapping strains were used to systematically compare distinct subsets of hepatocytes. During homeostasis, cells from both periportal zone 1 and pericentral zone 3 contracted in number, whereas cells from midlobular zone 2 expanded in number. These cells, which are sheltered from common injuries, also contributed to regeneration after pericentral and periportal injuries. Zone 2 repopulation was driven by the IGFBP2-mTOR-CCND1 axis. Hence, different regions of the lobule exhibit differences in their contributions to hepatocyte turnover, and zone 2 is the primary source of new hepatocytes during homeostasis and regeneration. Overall design: Primary hepatocytes were isolated with a two-step collagenase perfusion method described previously. Cells were washed and resuspended in hepatocyte washing medium (Life, 17704024). Single cell libraries were prepared using the 10x Genomics Chromium Single Cell 3' Reagents Kit v3. For each sample, 8,000 hepatocytes were loaded to target ~5,000 cells after recovery according to the manufacturer's protocol. Single cell libraries were generated and sequenced using the 150 bp paired-end Illumina NextSeq500 in the UTSW Children's Research Institute Sequencing Facility. 10X scRNA-seq data was preprocessed using the Cellranger v3.0.2 software. We used the mkfastq and count commands to process the 10x single-cell RNA-seq output into cells by gene expression count matrix, and used the aggr command to pool sequencing runs from 4 mice. All parameters were set to default except for the “force-cells” parameter, which was set to 4000.

肝脏以分区形式构建，不同分区的肝细胞（hepatocyte）表达不同的代谢酶。目前，肝脏再殖与再生过程中最关键的细胞类群仍未明确，原因是仅针对少数肝细胞亚群开展过命运图谱（fate-mapping）研究。本研究使用14种小鼠命运图谱品系，系统对比了不同的肝细胞亚群。在机体稳态状态下，门静脉周围1区和中央静脉周围3区的细胞数量均出现缩减，而肝小叶中间带2区的细胞数量则发生扩增。这类可免受常见损伤影响的细胞，同样在中央周区和门周区受损后参与了肝脏再生过程。2区的细胞再殖由IGFBP2-mTOR-CCND1信号轴驱动。因此，肝小叶不同区域对肝细胞更新的贡献存在差异，而2区是机体稳态及再生过程中新肝细胞的主要来源。

实验整体设计：采用此前报道的两步胶原酶灌注法分离原代肝细胞（primary hepatocyte）。将细胞洗涤后重悬于肝细胞洗涤培养基（购自Life Technologies，货号17704024）中。使用10x Genomics Chromium单细胞3'端试剂试剂盒v3构建单细胞文库。按照试剂盒说明书的操作流程，每份样本上样8000个肝细胞，以在回收后获得约5000个目标细胞。构建完成的单细胞文库在UTSW儿童研究所测序中心使用Illumina NextSeq500平台进行150bp双端测序。

使用Cell Ranger v3.0.2软件对10x单细胞RNA测序（scRNA-seq）数据进行预处理。通过mkfastq和count命令将10x单细胞RNA测序的原始数据处理为细胞-基因表达计数矩阵，并使用aggr命令将4只小鼠的测序数据进行合并。除"force-cells"参数被设置为4000外，其余参数均采用默认设置。