RNA-SEQ analysis of single cells extracted from the metanephric mesenchyme of E11.5 Crym-EGFP transgenic mice

We used micro-dissection with FACS sorting techniques to isolate single cells from the metanephric mesenchyme of the Embryonic day 11.5 (E11.5) developing kidney. A subset of these single cell populations is analysed individually via Fluidigm single cell analysis. This analysis will determine the transcriptional profile of each cell type, identify compartment specific transcripts, compartment specific transcript isoforms and cell-type specific long-noncoding RNAs. In addition the unbiased nature of RNA-SEQ will potentially identify novel transcripts that have not been annotated in the database. Kidneys are harvested from Tg(Crym-EGFP)GF82Gsat mice. Single cells are extracted from E11.5 metanephric mesenchyme using manual micro-dissection techniques. A subset of these cells is analyzed individually via Fluidigm single cell analysis. The long term goal is to generate a transcriptional atlas of the developing kidney.

本研究采用显微切割联合荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）技术，从胚胎发育第11.5天（E11.5）的发育中小鼠肾脏后肾间充质中分离单细胞。通过Fluidigm单细胞分析技术，对其中一部分单细胞群体进行单独检测。该检测可明确各细胞类型的转录组谱，鉴定区域特异性转录本、区域特异性转录本异构体以及细胞类型特异性长链非编码RNA。此外，RNA测序（RNA-seq）的无偏检测特性有望发现数据库中尚未注释的新型转录本。实验所用肾脏均取材自Tg(Crym-EGFP)GF82Gsat转基因小鼠。研究人员通过手动显微切割技术，从E11.5后肾间充质中分离单细胞，并对其中一部分细胞采用Fluidigm单细胞分析技术进行单独检测。本研究的长期目标为构建发育中小鼠肾脏的转录组图谱。