Figure 4.

Experimental protocol for library screening and analysis of contents of viral particles. A library of Sindbis virus replicons is electroporated into BHK-21 fibroblasts. Culture medium is changed after 16 h and left on the cells for 4 h to accumulate viral particles. This medium is used to infect cells. RNA is extracted 20 h post-infection. RT–PCR is performed using primers flanking the site of insertion of cDNA fragments. Amplicons are shotgun cloned in a bacterial plasmid. A macroarray of 1152 bacterial colonies is made. Inserts from 96 randomly picked colonies are sequenced. The most abundant inserts (between 5 and 10 clones) are labeled and hybridized to the macroarray. Inserts from negative colonies are sequenced.