Single-cells transcriptomes of mouse developing forelimbs

In order to try and evaluate Hoxd transcript heterogeneity during limb development, we produced single-limb cell transcriptomes of different origins, to see whether the apparently homogenous behavior in Hox gene transcriptional program as observed upon large-scale analyses was confirmed at the cellular level. We report here that Hoxd genes transcripts are present in various combinations in different limb cells and discuss the importance of these results in our understanding of how Hoxd genes are regulated and how their global functions are achieved in these structures. Single-cells transcriptomes from Hoxd11::GFP forelimbs with micro-dissection of autopod and zeugopod at embryonic day 12 (E12.5). Dissociated single-cells were obtained from eight Hoxd11::GFP forelimbs micro-dissected at E12.5. Cells with the highest level of GFP fluorescence (top 20%) were sorted using an Astrios cell sorter with a 100-um nozzle. 75bp reads were uniquely mapped to the latest Mus Musculus reference genome (mm10) and the ERCC sequences using bowtie2 (REF: doi:10.1038/nmeth.1923) in local mode.为探究并评估肢体发育过程中Hoxd基因转录本的异质性，我们制备了不同来源的单肢体细胞转录组，旨在验证大规模分析中观测到的Hox基因转录程序的表观均一性是否可在细胞水平得到证实。本文报道，Hoxd基因转录本以多种组合形式存在于不同的肢体细胞中，并讨论了这些结果对于理解Hoxd基因的调控机制及其在该结构中实现整体功能的重要意义。本数据集的单细胞转录组来自胚胎发育第12.5天（E12.5）的Hoxd11::GFP前肢，经显微解剖分为远侧肢段（autopod）与肢中段（zeugopod）。我们从8个该时期显微解剖得到的Hoxd11::GFP前肢中分离获得解离的单个细胞，使用Astrios细胞分选仪搭配100μm喷嘴，对GFP荧光强度最高的20%细胞进行分选。采用bowtie2（参考文献：doi:10.1038/nmeth.1923）的局部比对模式，将75bp测序读段唯一比对至最新的小家鼠（Mus musculus）参考基因组mm10及ERCC序列。