Small RNA-seq analysis of human airway epithelium infected by Human Bocavirus 1. Small RNA-seq analysis of human airway epithelium infected by Human Bocavirus 1

Human Bocavirus 1(HBoV1), which belongs to the genus Bocaparvovirus of the family Parvoviridae, infects well differentiated human airway epithelium which is at mitotically quiescent state. To systematicaly investigate the host and viral small RNA expression after HBoV1 infection of primary human airway epithelium cultured at an air-liquid interface (HAE-ALI), small RNA-seq was applied to study the small RNA transcriptome profile of HAE-ALI infected by HBoV1. Overall design: Primary human airway epithelial cells were cultured on collagen-coated, semipermeable membrane inserts of 0.33 cm2 and were polarized/differentiated at an ALI for 3-4 weeks. The well differentiated HAE-ALI cells were infected with HBoV1 at a multiplicity of infection (MOI) of 1,000 vgc/cell. At 7 days post infection, HBoV1 infected-cell were collected and RNA samples were prepared using the miRNeasy Mini Kit by following the manufacturer’s instruction. HAE cells from two Transwells were used as one RNA sample, totally 6 RNA samples from HBoV1 infected HAE-ALI cultures were performed for RNA isolation. All RNA samples were analyzed on an Agilent 2100 Bioanalyzer with an Agilent RNA 6000 Nano Kit to ensure an RNA Integrity Number (RIN) ≥ 8.0. Three RNA samples were chosen randomly for small RNA-seq.

人博卡病毒1型（Human Bocavirus 1, HBoV1）隶属于细小病毒科（Parvoviridae）博卡细小病毒属（Bocaparvovirus），可感染处于有丝分裂静止状态的分化成熟人气道上皮细胞。为系统解析人博卡病毒1型感染气液界面培养的原代人气道上皮细胞（HAE-ALI）后宿主与病毒的小RNA表达情况，本研究采用小RNA测序（small RNA-seq）技术，对HBoV1感染的HAE-ALI的小RNA转录组特征展开分析。
实验设计：将原代人气道上皮细胞接种于面积为0.33 cm²的胶原蛋白包被半透膜插入式培养板中，于气液界面（ALI）条件下极化分化3~4周。将分化成熟的HAE-ALI细胞以感染复数（multiplicity of infection, MOI）1000病毒基因组拷贝/细胞的剂量感染HBoV1。感染后第7天，收集HBoV1感染的细胞，按照试剂盒说明书操作，采用miRNeasy Mini Kit提取总RNA。以2个Transwell小室的HAE细胞作为1份RNA样本，共计从HBoV1感染的HAE-ALI培养物中制备6份RNA样本用于RNA提取。采用安捷伦2100生物分析仪（Agilent 2100 Bioanalyzer）搭配安捷伦RNA 6000 Nano试剂盒（Agilent RNA 6000 Nano Kit）对所有RNA样本进行质检，确保其RNA完整性指数（RNA Integrity Number, RIN）≥8.0。随机选取其中3份RNA样本进行小RNA测序。