Genome-wide CRISPR-CAS9 knockout library screening for rubella virus-entry factors in JEG3 cells. Homo sapiens strain: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).