RNAseq in WT and Dnmt3a KO HSCs naive and after 1-month of infection with M. avium

5000-10000 HSCs (KL CD150+ CD48- CD34-) were sorted into lysis buffer from the pools of naive or 1-month infected WT or Dnmt3a-/- mice (n=10-12 per group). RNA was isolated with the NucleoSpin ® RNA Plus XS kit (Macherey Nagel). RNA-seq libraries were prepared by using SMARTer® Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Takara Bio Usa). Illumina NovaSeq SP was used for sequencing with a paired-end sequencing length of 10bp. FASTQ files were preprocessed using HTS stream (https://github.com/ibest/HTStream) and the clean FASTQ file were aligned using STAR. Differential expression (DE) analysis of gene expression was performed using Limma-Voom. False discovery rate (FDR)<0.05 was considered statistically significant. We performed gene ontology analysis for differentially expressed genes with q value <0.05. Overall design: WT and Dnmt3a-/- HSCs extracted after 1 month of infection with M. avium or PBS (naïve)

将5000~10000个表型为KL CD150+ CD48- CD34-的造血干细胞（Hematopoietic Stem Cells，HSCs）从未感染（naïve）或经1个月鸟分枝杆菌（M. avium）感染的野生型（Wild Type，WT）及Dnmt3a基因敲除（Dnmt3a-/-）小鼠的细胞混合池中分选出来，置于裂解缓冲液中，每组设置10~12只小鼠重复。
使用NucleoSpin® RNA Plus XS试剂盒（Macherey Nagel）提取总RNA。
采用SMARTer® Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian试剂盒（Takara Bio美国分公司）构建RNA测序文库。
使用Illumina NovaSeq SP测序平台进行双端测序，测序读长为10 bp。
利用HTS stream（https://github.com/ibest/HTStream）对FASTQ格式文件进行预处理，并通过STAR软件将质控后的干净FASTQ序列进行基因组比对。
采用Limma-Voom软件开展基因表达差异分析（Differential Expression，DE）。
以错误发现率（False Discovery Rate，FDR）<0.05作为统计学显著性阈值。
针对q值<0.05的差异表达基因进行基因本体（Gene Ontology，GO）功能富集分析。
实验整体设计：分别从经鸟分枝杆菌感染1个月或磷酸盐缓冲液（PBS，即未感染对照组）处理的小鼠中提取野生型（WT）及Dnmt3a-/-造血干细胞。