Transcriptome profiling of cytoplasmic fraction of naive CD8 T cells from Nxf1 mutant and control mice [Lymphocytes]

Purpose: Nxf1 is thought to be an essential nuclear exporter of messenger RNA (mRNA) in eukaryotic cells. Whether perturbations in the Nxf1 pathway affect mammalian physiology is not known. The aim of this study is to determine the impact of a Nxf1 mutation in the representation of mRNA transcripts in the cytoplasm of naive CD8 T cells. Methods: For naive CD8 T cell isolation, splenocyte suspensions were submitted to red blood cell lysis, stained first with biotinylated CD8 antibodies and then labelled with anti-Biotin microbeads. CD8+ T cells were magnetically enriched by positive selection on MACS separation columns, stained with fluorescently-labelled antibodies before being FACS-sorted as CD8+ CD44low CD62L+. Sorted naive T cells were fractionated with Norgen cytoplasmic and nuclear fractionation RNA purification kit. Only cytoplasmic fractions were used for subsequent RNAseq. CD8 naive T cells were FACS-sorted from individual Nxf1 mutant males (n=3) and littermate wild-type controls (n=3) .研究目的：Nxf1被认为是真核细胞中信使RNA（mRNA）的关键核输出蛋白。目前尚不明确Nxf1通路的扰动是否会影响哺乳动物的生理机能。本研究旨在探究Nxf1突变对初始CD8 T细胞胞质内mRNA转录本表达特征的影响。 实验方法：分离初始CD8 T细胞时，先对脾细胞悬液进行红细胞裂解，先用生物素标记的CD8抗体染色，再用抗生物素微珠进行标记。通过磁激活细胞分选（Magnetic Activating Cell Sorting, MACS）分选柱的阳性磁选步骤富集CD8+ T细胞，随后用荧光标记抗体染色，以CD8+ CD44low CD62L+的表型进行荧光激活细胞分选（Fluorescence Activated Cell Sorting, FACS）。将分选得到的初始T细胞使用Norgen胞质与核组分RNA纯化试剂盒进行组分分离，仅选取胞质组分用于后续的RNA测序（RNAseq）。本实验从3只Nxf1突变雄性小鼠及3只同窝野生型对照小鼠中分别流式分选CD8初始T细胞。