Transcription profile of intracellular H37Ra of bEnd.3 and Raw 264.7 cells

Transcription profile of intracellular H37Ra of bEnd.3 and Raw 264.7 cells 3 d.p.i Raw 264.7 cells and bEnd.3 cells were infected with M.tb (MOI=10) for 6 h at 37°C in 5% CO2. Infected cells were lysed with 30 mL of GTC buffer at day 3 postinfection and centrifuged for 20 min at 5,000 g to pellet bacteria. The bacterial pellets were resuspended in 1 mL of GTC and centrifuged for 2 min at 15,000 g. The bacterial pellets were disturbed in 1mL of Trizol reagent by vortex for 10 min. Total RNAs were extracted as described above. rRNA was removed under the guidelines of Ribo-Zero rRNA Removal Kit (Illumina). The mRNAs were reverse-transcripted to cDNA and converted into double-stranded cDNA molecules. Following end-repair and dA-tailing, the paried-end sequencing adaptors were ligated to the ends of the cDNA fragments, and then subjected to library amplification and purification. The purified libraries were validated and quantified using the Agilent 2100 Bioanalyzer (Agilent Technologies) and sequenced with Hi-Seq 10× instrument

bEnd.3与Raw 264.7细胞内的结核分枝杆菌H37Ra株感染后第3天的转录谱。将Raw 264.7与bEnd.3细胞以感染复数（Multiplicity of Infection, MOI）=10的比例接种结核分枝杆菌（Mycobacterium tuberculosis, M.tb），于37℃、5% CO₂环境中孵育6小时。于感染后第3天，使用30 mL GTC缓冲液裂解感染细胞，以5000×g离心20分钟收集细菌沉淀。将所得细菌沉淀重悬于1 mL GTC缓冲液中，以15000×g离心2分钟。随后将细菌沉淀重悬于1 mL Trizol试剂内，涡旋震荡10分钟以充分裂解。按照前述操作提取总RNA，使用Ribo-Zero核糖体RNA去除试剂盒（Illumina公司）按照说明书去除核糖体RNA（rRNA）。将信使RNA（mRNA）反转录为互补DNA（cDNA）并制备为双链cDNA分子。对cDNA片段进行末端修复及dA尾加尾后，于其末端连接双端测序接头，随后进行文库扩增与纯化。使用安捷伦2100生物分析仪（Agilent Technologies公司）对纯化后的文库进行质检与定量，最终采用HiSeq 10×测序仪完成测序。