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High-throughput RNA interference screening using pooled shRNA libraries and next generation sequencing

David Sims, Ana M Mendes-Pereira, Jessica Frankum, Darren Burgess, Maria-Antonietta Cerone, Cristina Lombardelli, Costas Mitsopoulos, Jarle Hakas, Nirupa Murugaesu, Clare M Isacke, Kerry Fenwick, Ioannis Assiotis, Iwanka Kozarewa, Marketa Zvelebil, Alan Ashworth* and Christopher J Lord*

Author Affiliations

The Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB, UK

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Genome Biology 2011, 12:R104  doi:10.1186/gb-2011-12-10-r104

Published: 21 October 2011

Additional files

Additional file 1:

Engineered depletion of shRNAs. To establish the sensitivity of the screening system, we performed a series of engineered depletion experiments. We manually altered the representation of constructs in a 10,000 shRNA screening pool so that approximately 1,000 hairpins were depleted by 75%, approximately 1,000 depleted by 50% and approximately 1,000 depleted by 25%.

Format: EPS Size: 836KB Download file

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Additional file 2:

Detection of hairpin depletion at reduced read counts. Reads were sampled at random from an engineered depletion experiment involving approximately 10 million reads to give datasets of either approximately 5 million or approximately 2.5 million reads in total. shRNA depletion was estimated from these new datasets to show that depletion of 50% could be observed in datasets containing approximately 2.5 million reads.

Format: EPS Size: 1023KB Download file

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Additional file 3:

Positive and negative controls for the MCF7 viability screen were established using a single hairpin GFP-competition assay. The bar chart indicates the proportion of GFP positive cells remaining after 2 weeks of culture. The bar represents the average from three biological replicates. The error bars indicate the standard deviation.

Format: EPS Size: 8.3MB Download file

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Additional file 4:

Detailed shRNA screening protocols. This Word document describes in detail all of the steps of the shRNA screening protocol from library generation to massively parallel sequencing.

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