High-throughput high-multiplexed screening for splicing factors

?An arrayed library containing pooled siRNAs targeting 18,480 human genes ?(Dharmacon, Lafayette, CO) was screened as previously described (Roosing et al., 2015).?Briefly, 384-well plates with optical bottom (Greiner Bio-One, Monroe, NC) were spotted ?with 1 µl of 0.5 µM siRNA. Reverse transfection was next performed using Lipofectamine ?RNAiMAX at the final siRNA concentration of 10 nM. Culture medium was replaced with ?DMEM 24 hrs after transfection and cells were incubated for additional 48 hrs before lysis ?in SDS buffer for downstream analysis. ?These siRNAs were distributed in 57 plates, and after screen, we pooled ?4 bar-coded plates for sequencing per Illumina sequencing lane. Overall design: Identification and characterization of splicing factors in human genome.