mRNA Sequencing
To detect potential changes in host cell gene expression patterns after
transfection, parallel cultures of PC-3, Jurkat, and primary T cells
were passaged into fresh plates or flasks. Control (untransfected)
cultures were left to grow for 48 hours, while the remaining cultures
were transfected with pEF-GFP one day after passaging. On the following
day, a small fraction (10%) of each culture was then analyzed using
flow cytometry and fluorescent microscopy to confirm EGFP expression,
then total RNA samples were extracted from the remaining cells with a
Qiagen RNEasy kit. The RNA samples were then submitted to the Beijing
Genomics Institute (BGI, Hong Kong, China) for library preparation,
next-generation sequencing, and data analysis to obtain the FPKM values
shown in Tables 1-3.