RNA-SEQ analysis of various developing and adult kidney components

We used micro-dissection techniques and/or FACS to isolate cell types from the developing and adult kidney (E11.5 ureteric buds, E12.5, P1 and P4 cap mesenchyme, E15.5 collecting ducts, proximal tubules, ureter, adult renal proximal tubules, podocytes, endothelial and mesangial cells). RNA-SEQ analysis was performed to determine the transcriptional profile of each cell type, identify component specific transcripts and 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. Total RNA is obtained from micro-dissected and/or FACS isolated embryonic and adult kidney components. The long term goal is to generate a transcriptional atlas of developing kidney.我们采用显微解剖技术（micro-dissection techniques）和/或荧光激活细胞分选（FACS），从发育中和成年肾脏中分离各类细胞类型，包括胚胎11.5天（E11.5）的输尿管芽（ureteric buds）、胚胎12.5天（E12.5）、出生后1天（P1）与出生后4天（P4）的帽状间充质（cap mesenchyme），胚胎15.5天（E15.5）的集合管（collecting ducts）、近端小管（proximal tubules）、输尿管，以及成年肾脏的近端小管、足细胞（podocytes）、内皮细胞（endothelial cells）与系膜细胞（mesangial cells）。随后开展RNA测序（RNA-Seq）分析，以解析各类细胞的转录谱，鉴定细胞组分特异性的转录本、剪接异构体，以及细胞类型特异性的长链非编码RNA（long-noncoding RNAs）。此外，RNA测序的无偏倚特性还有望发掘数据库中尚未注释的新型转录本。本研究的总RNA均提取自经显微解剖或FACS分离的胚胎与成年肾脏组分。本研究的长期目标为构建发育中肾脏的完整转录组图谱。