| Description |
Five fusion genes cDNA sequences were synthesized and constructed into lentiviral vectors respectively. To construct a cell line with stable expression of fusion genes, every lentiviral vector and two auxiliary packaging plasmids were co-transfected into the 293T cells. After 48 hours, the supernatant was collected from the 293T cells and filtered through a 0.45 uM membrane. The five different recombinant lentiviral particles containing the target fusion genes and the green fluorescent protein (GFP) reporter gene were collected. Then, the five recombinant lentivirus particles infected into 293T cells, respectively. 72 hours after infection, the medium was changed and the expression of GFP in cells was checked under a fluorescence microscope for determining if lentivirus infection was successful. After the infection, five cell lines expressing the target fusion genes were collected. The single GFP positive cells were filtered into a 96-well PCR plate by fluorescence activated cell sorting (FACS) for the single-cell RNA library construction. Poly(A)-transcripts of total RNA of single cells were reverse transcribed and amplified using the SMART-seq2(7, 8) protocol. The amplified cDNA was tagmented by Nextera XT kit (Illumina) and libraries were sequenced by NovaSeq (Illumina). The 293T cells were purchased from the National Infrastructure of Cell Line Resource |
