Genome-wide CRISPR-CAS9 knockout library screening for rubella virus-entry factors in JEG3 cells

Human choriocarcinoma JEG3 cells (ATCC HTB-36) were infected with lentiviral pools of human GeCKOv2 CRISPR knockout pooled library (Addgene pooled library #1000000048, #1000000049). One hundred million of JEG3-GeCKOv2-Lib cells were inoculated with the Rubella virus HPV-77 strain at an MOI of 10. The cells were incubated at 35°C for approximately 7 days.The surviving cells were passaged, inoculated with Rubella virus again and further incubated for 7 days. The genomic DNA was extracted from ten million of the original library cells (control cells) or the selected cells. These screenings were performed in duplicate. Amplicons containing sgRNA integrants from two control cells and two selected cells were prepared by 10 forward primers with different lengths of spacer nucleotides and one each of the four reverse primers with different index sequences using PrimeSTAR GXL DNA Polymerase (Takara Bio, Kusatsu, Japan). 4 nM of Purified PCR products from four different templates was mixed and applied for deep sequencing using MiSeq Reagent Kit v3 (150 cycles) (Illumina, San Diego, CA) in the MiSeq system (Illumina).

人类绒毛膜癌JEG3细胞（ATCC HTB-36）经人类GeCKOv2 CRISPR敲除混合文库的慢病毒池感染（Addgene混合文库编号#1000000048、#1000000049）。取1亿个JEG3-GeCKOv2-Lib细胞，以感染复数（multiplicity of infection, MOI）10接种风疹病毒HPV-77株，于35℃培养约7天。将存活细胞传代后再次接种风疹病毒，继续培养7天。分别从1000万个原始文库细胞（对照组细胞）及筛选得到的细胞中提取基因组DNA。本筛选实验设置两个生物学重复。使用PrimeSTAR GXL DNA聚合酶（Takara Bio，日本滋贺县），以10条带有不同长度间隔核苷酸的正向引物以及4条各携带不同索引序列的反向引物，对两个对照组细胞和两个筛选组细胞的样本进行PCR扩增，制备携带单导RNA（single guide RNA, sgRNA）整合序列的扩增子。将来自4种不同模板的纯化PCR产物以4 nM浓度混合后，使用MiSeq测序系统（Illumina，美国加利福尼亚州圣地亚哥）搭配MiSeq Reagent Kit v3（150个循环）（Illumina）进行深度测序。