ON-TARGETplus Human ANO1 (55107) siRNA - SMARTpool

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Seed 6000 cells/well	siRNA concentration-50 nM After 24h , exchange to phenol red-free DMEM containing 1.25 µg/ml doxycycline	Analyse 72 h post-transfection

Upstream tips
Seed 6000 cells/well
Protocol tips
siRNA concentration-50 nM After 24h , exchange to phenol red-free DMEM containing 1.25 µg/ml doxycycline
Downstream tips
Analyse 72 h post-transfection

Publication protocol

The screen was performed at the FMP Screening Unit using the Ambion Silencer® Human Genome siRNA Library V3 (Life Technologies) containing 189 384-well plates. This library targets each gene by three independently placed siRNAs. The screen was performed in two replicates. Each screening plate contained several controls like siRNA pools against YFP (Silencer GFP siRNA from Ambion), a non-targeting siRNA (Silencer Negative Control from Ambion), an siRNA pool against AE2 (ON-TARGETplus SMARTpool siRNA SLC4A2 from Thermo Scientific) and a cell death-inducing siRNA mixture (AllStars Hs Cell Death Control siRNA from Qiagen). For detailed plate layout see fig. S2. For siRNA transfection, in each
well of the 384-well assay-plate 8 µl of a 500 nM library-siRNA-OptiMEM® solution was mixed
with 0.2 µl Lipofectamine® RNAimax transfection reagent (Life Technologies) previously diluted in 11.8 µl Opti-MEM® (Life Technologies). Subsequently 6000 cells/well in antibioticfree DMEM were seeded onto the pre-dispensed transfection mixture using a BioTek EL406TM dispenser resulting in a final concentration of 50 nM siRNA in a total volume of 80 µl per well. After 24 h the cell culture medium was exchanged to phenol red-free DMEM containing 1.25 µg/ml doxycycline to induce YFP-expression.

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