Small non-coding RNA expression in developing mouse nephron progenitors

The overall goal of this study is to determine the small non-coding RNA expression profile in developing mouse nephron progenitors and whole kidney. Using a limited digestion and negative selection approach, an enriched fraction of nephron progenitors were isolated from E15.5 whole kidney samples followed by small RNA-Sequencing (sRNA-Seq). A total of 3 biological replicates of mouse nephron progenitors and whole kidney samples were used for the sRNA-Seq. The NEBNext Multiplex Small RNA Library Prep Kit allowed us to prepare sequencing libraries from as little as 100ng total RNA. Multiplex sequencing was performed using the Illumina NextSeq 550 system with 50bp single reads, resulting in approximately 18 million reads per sample. Reads were aligned to the mm10 genome using Bowtie2, and the miRDeep2 software package was used to identify and quantify known and novel micro RNA (miRNA) within our sRNA-seq libraries. Differential expression analysis of miRNA expression between nephron progenitor and whole kidney samples identified 162 differentially expressed miRNAs (padj <= 0.05). 3 biological replicates of nephron progenitors and whole kidney samples were used for the sRNA-Seq. Each replicate consists of material pooled from a single litter of embryos.本研究的总体目标为解析发育中小鼠肾单位祖细胞及全肾脏中的小型非编码RNA（small non-coding RNA）表达谱。本研究采用有限酶切与阴性筛选策略，从胚胎15.5天（E15.5）的全肾脏样本中分离得到富集的肾单位祖细胞组分，随后开展小型RNA测序（small RNA-Sequencing，下称sRNA-Seq）。本研究共使用3份小鼠肾单位祖细胞与全肾脏样本的生物学重复开展sRNA-Seq实验。本次实验采用NEBNext Multiplex Small RNA Library Prep Kit试剂盒，仅需100ng总RNA即可完成测序文库的构建。测序工作借助Illumina NextSeq 550测序系统完成，采用50bp单端读长的多重测序模式，每份样本平均产出约1800万条读段。使用Bowtie2将测序读段比对至mm10参考基因组，并通过miRDeep2软件包识别并定量sRNA-Seq文库中的已知及新型微RNA（microRNA，下称miRNA）。对肾单位祖细胞与全肾脏样本间的miRNA表达开展差异表达分析，最终筛选得到162个差异表达miRNA（padj ≤ 0.05）。本研究共使用3份肾单位祖细胞与全肾脏样本的生物学重复开展sRNA-Seq实验，每份重复样本均由同一窝胚胎的材料混合制备而成。