RSV and HBoV1 co-infection causes more severe pathogenic damage to human respiratory epithelium than single infection

To analyze the differences in HAE gene expression between RSV and HBoV1 co-infection and single infection, total RNA of HAE with “RSV single”, “HBoV1 single”, “RSV and HBoV1” infection at 0 (mock-), 2 and 6 d.p.i. was isolated using the Magzol Reagent (Magen, China) according to the manufacturer's protocol. The quantity and integrity of RNA yield was assessed by using the K5500 (Beijing Kaiao, China) and the Agilent 2200 TapeStation (Agilent Technologies, USA) separately. the mRNA was enriched by oligo dT according to instructions of NEB Next® Poly(A) mRNA Magnetic Isolation Module (NEB,USA), and then fragmented to approximately 200 bp. Subsequently, the RNA fragments were subjected to first strand and second strand cDNA synthesis followed by adaptor ligation and enrichment with a low-cycle according to instructions of NEBNext® Ultra™ RNA Library Prep Kit for Illumina. The purified library products were evaluated using the Agilent 2200 TapeStation and Qubit (Thermo Fisher Scientific, USA). The libraries were sequenced by Illumina (Illumina, USA) with paired-end 150bp.为分析呼吸道合胞病毒（RSV）与人类博卡病毒1型（HBoV1）共感染及单一感染后人气道上皮（HAE）的基因表达差异，本研究依照制造商操作规程，采用Magzol试剂（Magen，中国）提取感染后0（空白对照，mock-）、2、6天的"单纯RSV感染""单纯HBoV1感染""RSV与HBoV1共感染"样本的HAE总RNA。分别使用K5500分光光度计（北京凯奥，中国）与安捷伦2200 TapeStation系统（安捷伦科技，美国），对提取所得RNA的浓度与完整性分别进行评估。随后参照NEB Next® Poly(A) mRNA磁珠分离试剂盒（NEB，美国）的操作说明，通过寡聚dT磁珠富集mRNA，并将其片段化至约200 bp。继而依据适配Illumina测序平台的NEBNext® Ultra™ RNA文库制备试剂盒（NEB，美国）的实验流程，对RNA片段进行第一链与第二链cDNA合成，随后完成接头连接与低循环数富集扩增。纯化后的文库产物采用安捷伦2200 TapeStation系统与Qubit荧光定量仪（赛默飞世尔科技，美国）进行质检。最终采用Illumina测序平台（Illumina，美国）对文库开展150 bp双端测序。