Identification of required host factors for SARS-CoV-2 infection in human cells [RNA-seq]. Identification of required host factors for SARS-CoV-2 infection in human cells [RNA-seq]

RNA-seq was performed to determine gene expression (RNA) levels and SARS-CoV-2 viral reads in A549-ACE2 cells treated with DMSO or Amlodipine. Overall design: ACE2-A549s cells were treated with either DMSO or amlodipine (Millipore Sigma, Cat #A5605) resuspended in DMSO one hour before infection. Amlodipine was used at a concentration of 10uM. A549-ACE2 cells were infected at MOI of 0.1 with SARS-CoV-2 for 24 hours before harvest. RNA-seq libraries were built from 1ug of RNA per sample using the TruSeq Stranded mRNA Library Prep Kit (Illumina) according to the manufacturer’s instructions. Sequencing libraries were sequenced using an Illumina NextSeq 500 platform and fastq files were generated using bcl2fastq (Illumina). Reads were aligned to hg19 using STAR aligner via the RNA-Seq Alignment application (Basespace, Illumina). Alignments to the SARS-CoV-2 genome (GenBank: MN985325.1) were determined using bowtie (Robinson et al., 2010).

本研究通过RNA测序（RNA-seq）技术，检测经二甲基亚砜（DMSO）或氨氯地平处理的A549-ACE2细胞中的基因表达（RNA）水平与严重急性呼吸综合征冠状病毒2（SARS-CoV-2）病毒读段。
总体实验设计：感染前1小时，将A549-ACE2细胞分别用二甲基亚砜（DMSO）或氨氯地平（以DMSO为溶剂重悬，购自密理博西格玛（Millipore Sigma），货号A5605）处理，氨氯地平的终浓度为10μM。随后以感染复数（MOI）为0.1的比例，用SARS-CoV-2感染A549-ACE2细胞，感染24小时后收取细胞样本。
每份样本取1μg总RNA，按照制造商操作说明，使用Illumina的TruSeq链特异性mRNA文库制备试剂盒构建RNA测序文库。利用Illumina NextSeq 500测序平台对构建好的测序文库进行测序，并通过bcl2fastq（Illumina）工具生成FASTQ格式文件。借助Illumina Basespace平台的RNA测序比对应用，使用STAR比对工具将测序读段比对至人类参考基因组hg19。针对SARS-CoV-2基因组（GenBank登录号：MN985325.1）的序列比对分析通过Bowtie工具完成（Robinson等，2010）。