RNA-seq of nulliparus Robo1+/+ and Robo1-/- luminal progenitors

The goal of the study was to compare gene expression of Robo1+/+ and Robo1-/- luminal progenitors. Total RNAs were then extracted from FACS purified luminal progenitor cells, harvested from Robo1+/+ or Robo1-/- mice (n=3 per genotype, two animals per n) using TRIreagent LS (Sigma, T3934). Poly(A)+ RNA sequencing libraries were made from each sample using the TruSeq RNA library preparation kit v.1 (Illumina). Illumina RNA PolyA library preparation guide. A total of 6 libraries were created by PCR amplification with Illumina barcoding primers using kit recommended conditions and quantified using a Bioanalyzer DNA 1000 kit (Agilent). Overall design: Total RNAs were isolated from FACS purified luminal progenitors, and sequenced on a HiSeq 2000 Sequencing system (Illumina).

本研究旨在对比Robo1野生型（Robo1+/+）与敲除型（Robo1-/-）小鼠的腔系祖细胞的基因表达差异。随后，使用TRIreagent LS（Sigma，货号T3934），从经荧光激活细胞分选（Fluorescence-Activated Cell Sorting，FACS）纯化的腔系祖细胞中提取总RNA，这些细胞采自Robo1+/+或Robo1-/-小鼠，每种基因型设置3个生物学重复，每个重复对应2只实验动物。采用TruSeq RNA文库制备试剂盒v.1（Illumina），为每一份样本构建Poly(A)+ RNA测序文库，并严格遵循Illumina官方RNA PolyA文库制备指南。使用Illumina带条形码引物，按照试剂盒推荐的反应条件进行PCR扩增，共构建得到6个测序文库；随后采用Agilent Bioanalyzer DNA 1000试剂盒对各文库进行定量检测。实验整体设计：从经FACS纯化的腔系祖细胞中分离总RNA，随后在HiSeq 2000测序系统（Illumina）上完成测序。