Deep RNA Sequencing of native mouse podocytes and non-podocytes glomerular cells

Purpose: Next-generation sequencing (NGS) was used to define the transcriptome of native mouse podocytes and non-podocytes glomerular cells as part of a project aiming to define the molecular fingerprint of mouse podocytes. Method: Glomeruli from 29 Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J x hNPHS2Cre mice at the age of 10 weeks were purified and a single cell solution was prepared to seperate GFP-expressing (podocytes) and GFP-negative (non-podocytes glomerular cells) cells by FACS sorting. RNA was extracted and prepared for further analysis using directional, polyA+ library preparation. An Illumina HiSeq2500 was used for a paired-end sequencing of 100 cycles . Salmon and Sleuth were used for downstream analysis. Results: A total of 100 Million reads each from podocytes and non-podocytes glomerular cells could be used for further analysis.目的：本研究作为明确小鼠足细胞分子特征图谱项目的组成部分，采用下一代测序（Next-generation sequencing, NGS）技术解析天然小鼠足细胞及非足细胞肾小球细胞的转录组。 方法：选取29只10周龄的Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J × hNPHS2Cre小鼠的肾小球进行纯化，制备单细胞悬液，通过荧光激活细胞分选（FACS, Fluorescence-Activated Cell Sorting）分离表达绿色荧光蛋白（GFP, Green Fluorescent Protein）的足细胞与GFP阴性的非足细胞肾小球细胞。提取总RNA后，采用定向polyA+文库制备方案完成文库构建，用于后续分析。使用Illumina HiSeq2500平台开展100个循环的双端测序。下游数据分析采用Salmon与Sleuth工具完成。 结果：足细胞与非足细胞肾小球细胞各获得总计1亿条有效读数，均可用于后续分析。