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

Human choriocarcinoma JAR cells (ATCC HTB-144) were infected with lentiviral pools of human GeCKOv2 CRISPR knockout pooled library (Addgene pooled library #1000000048, #1000000049). One hundred million of JAR-GeCKOv2-Lib cells were inoculated with the Rubella virus Cendhill strain at an MOI of 10. The cells were incubated with medium containing 1 µM ABT-737 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).