Figure 1. The multiplexed RNAi screening approaches.

U87 cells were transduced with the lentivirus library. Following antibiotic selection, the cells were used for migration assay using a Matrigel invasion chamber. In approach 1, migrated and non-migrated cells were separately collected for genomic DNA extraction. The barcode region was amplified by PCR and labeled with CY3 or CY5, and used for microarray analysis to compare the shRNA abundance in either population. In approach 2, the migrated cells were collected and amplified, then subjected to another round of migration selection. The procedure was repeated 5 times before the final cells were used for single cell amplification in 96-well plates. After clonal expansion, genomic DNA was extracted and sequenced to determine the shRNA sequences in each clone.