Method
High-throughput RNA interference screening using pooled shRNA libraries and next generation sequencing
The Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB, UK
Genome Biology 2011, 12:R104 doi:10.1186/gb-2011-12-10-r104
Published: 21 October 2011Additional 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
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
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
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.
Format: DOC Size: 845KB Download file
This file can be viewed with: Microsoft Word Viewer
