Large-Scale RNA Interference Screening in Mammalian Cells Identifies Novel Regulators of Mutant Huntingtin Aggregation
Figure 2
Summary of the shRNA screening.
Total 11346 shRNA clones were subjected to the first screening using Nhtt150Q-EGFP cells. The screening was performed in duplicate (partly in triplicate), and shRNAs altering Nhtt150Q-EGFP aggregation at <−1.5 or >1.5 of z-score were selected as positive candidates. Obtained 602 clones were then subjected to the second screening using Nhtt16Q-EGFP cells to exclude the shRNAs modifying the aggregation by altering Nhtt expression itself. shRNAs altering EGFP intensity by 15% compared with the control were removed. Remaining 270 candidates were subjected to the third screening using Nhtt150Q-EGFP cells for confirmation (triplicate). We performed statistical analysis (t-test) in the third screening and shRNAs with P<0.1 were included as candidates. Finally, 111 shRNAs were obtained, among which 63 shRNAs suppressed and 48 shRNAs enhanced the Nhtt150Q-EGFP aggregation.