Transcriptome analysis of mouse olfactory sensory neurons expressing 3 different types of olfactory receptor

The goal of RNA-seq is to explore whether olfactory sensory neurons (OSNs) that display a specific olfactory receptor (OR) express a distinct transcriptional program from OSNs that display a different OR. The GFP knockin mice (Olfr73-IRES-GFP, Olfr1507-IRES-GFP, and Olfr160-IRES-mCherry) was used to label the OSN expressing specific OR. These cells was then isolate with fluorescence activated cell sorting. For each sample, at least 5000 cells are collected, there biological replicates were collected for each genotype and in total 9 samples were prepared for RNA-seq libraries with using NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina® (NEB) following the manufacturer's guidelines with one alteration, which was to increase the insert length to ~300 base pair. Libraries were sequenced using paired end 150 cycle reads on an Illumina Hiseq4000 (Novogene). The sequencing reads were processed using the DolphinNext RNAseq pipeline (https://dolphinnext.umassmed.edu/index.php?np=1&id=732). The default settings were employed except STAR v2.6.1 and RSEM v1.3.1 were used for alignment and quantification, respectively. Differential analysis were conducted in DESeq2. The significant genes were deducted by computing Wald test with corrected p < 0.05. Our results shows that different types of OSNs express significantly different patterns of genes, especially the genes involved in axonal guidance. Messenger RNA from olfactory sensory neuron expressing different olfactory receptor (Olfr73, Olfr1507, or Olfr160) were extracted and cDNA libraries were generated for deep sequencing, in duplicate, using Hiseq4000本RNA测序（RNA-seq）实验旨在探究表达特定嗅觉受体（olfactory receptor, OR）的嗅觉感觉神经元（olfactory sensory neurons, OSNs），是否与表达其他嗅觉受体的嗅觉感觉神经元存在独特的转录程序差异。本研究使用GFP敲入小鼠（Olfr73-IRES-GFP、Olfr1507-IRES-GFP及Olfr160-IRES-mCherry）对表达特定嗅觉受体的嗅觉感觉神经元进行标记，随后通过荧光激活细胞分选分离目标细胞。每个样本至少收集5000个细胞，每种基因型设置3次生物学重复；总计制备9个RNA测序文库，文库构建采用适配Illumina®平台的NEBNext® 单细胞/低起始量RNA文库制备试剂盒（NEB），严格遵循制造商操作指南，仅调整一项参数：将插入片段长度调整至约300碱基对。文库在Illumina Hiseq4000平台上采用150bp双端测序模式进行测序，测序服务由诺禾致源（Novogene）提供。测序下机数据通过DolphinNext RNAseq分析流程（https://dolphinnext.umassmed.edu/index.php?np=1&id=732）进行处理，分析采用默认参数，仅将序列比对软件替换为STAR v2.6.1，基因表达定量软件替换为RSEM v1.3.1。差异表达分析通过DESeq2工具完成，通过Wald检验计算并经校正后P值<0.05的基因被判定为差异显著基因。研究结果显示，不同类型的嗅觉感觉神经元的基因表达模式存在显著差异，尤其在轴突导向相关基因中表现突出。本实验提取表达不同嗅觉受体（Olfr73、Olfr1507及Olfr160）的嗅觉感觉神经元的信使RNA，构建cDNA文库进行深度测序，设置2次生物学重复，测序平台为Illumina Hiseq4000。