the den2-s2 virus used in the primary screen was isolated via serial passage of den2-ngc ( a gift from dr . a. de silva  unc - ch ) in d.mel-2 cells ( invitrogen ) . the experimental schedule for the primary and secondary screens is outlined in fig . the human screen was done with huh-7 cells treated independently with two sirnas ( qiagen ) for each gene product and den2-ngc according to the schedule outlined in fig . infected cells were labeled with the anti - e , 4g2 primary antibody ( isolated from the di-4g2 - 4 - 15 hybridoma ( atcc#hb-112 ) ) and alexa-488 anti - mouse secondary ( invitrogen ) , and counterstained with hoescht 33342 ( sigma ) . imaging and analysis of infection was done with a cellomics arrayscan vti hcs machine . selection of candidates from the primary screen was done using a nonparametric approach , the sum rank algorithm , in order to produce an appropriate summary statistic of each dsrna tested in duplicate . briefly , within each plate , wells were ranked by the percentage of infected cells , with the well with the lowest percentage infected cells given rank = 1 . for each dsrna tested in duplicate , we calculated a sum rank ( sr ) statistic using the formula : sr = rank on plate # 1 + rank on plate # 2 . the number of times a given sr is expected to occur near the lower extreme ( sr = 2 ) for a single pair of duplicate plates is given by : e[sr ] = ( sr - 1 ) / ( # valid wells ) . srs were calculated for every dsrna in the drosophila genome , with e[sr ] scores below 0.065 used to select potential targets ( 218 dsrnas ) for further analysis ( see supplementary fig .