Cells were washed with warm PBS, trypsinized, resuspended in warm medium, and counted. An aliquot of the 80k human shRNA lentivirus pool and either polybrene (4–8 g/mL) or protamine sulfate (5 g/mL) were added such that an MOI of 0.3 to 0.4 would be achieved (determined by prior testing of each cell line). Cell–lentivirus mixtures were plated into 15-cm-diameter culture dishes and incubated at 37°C with 5% CO2. Twenty-four hours postinfection, the medium was replaced with fresh medium containing puromycin (puromycin concentration determined by prior tests on each cell line), and cells were incubated for an additional 48 hours. Culture dishes were washed with warm PBS to remove dead cells, and surviving cells were collected by trypsinization and resuspended in warm medium. Cell populations were quantified, aliquots of 2 x 10^7 cells were removed and pelleted by centrifugation, and 3 replicate populations of 2 x 10^7 cells were plated at appropriate density into 15-cm-diameter culture dishes. When replicate populations reached 80% to 90% confluency, cells from each replicate were collected by trypsinization and mixed (cells from different replicates were not comingled). From these mixtures, 2 aliquots of 2 x 10^7 cells were removed, pelleted, and frozen down, while one aliquot of 2 x 10^7 cells was replated for further growth. This step was repeated until a minimum of 6 to 8 doublings for each replicate cell population was obtained. (see PMID: 22585861)
Growth protocol
Each cell line was grown to a population size of ~2 x 10^8 cells in the appropriate medium (see PMID: 22585861)
Extracted molecule
genomic DNA
Extraction protocol
Genomic DNA was prepared from cell pellets using the QIAmp Blood Maxi kit (Qiagen #51194). Genomic DNA was precipitated using ethanol and sodium chloride and resuspended at 400 ng/L in 10 mmol/L Tris-HCl, pH 7.5. shRNA populations from cell lines were amplified via PCR and prepared and applied to GMAP arrays. (see PMID: 22585861)
Label
biotin
Label protocol
Affymetrix protocol
Hybridization protocol
Hybridization solutions consisted of 2 μg of probe for 78 k shRNA pools (2.3 μg for 90 k shRNA pools) in buffer containing 1x MES, 0.89M NaCl, 20 mM EDTA, 0.0001% Tween 20, 0.5 mg/ml BSA, 0.1 mg/ml herring sperm DNA, 0.05 nM biotinylated B2 oligo (Affymetrix), 10% DMSO, 20 μM each blocking oligos (Block_1 5'-AATGGACTATCATATGCTTACCGTAACTTGAA-3', Block_2 5'-TTCAAGTTACGGTAAGCATATGATAGTCCATT-3', Block_3 5'-GTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCG-3', Block_4 5'-CGGTGTTTCGTCCTTTCCACAAGATATATAAAGCCAAGAAATCGAAATAC-3'), and sterile water to a final volume of 138 μl. Samples in buffer were denatured at 95°C for 10 minutes, incubated at 40°C for 5 minutes, collected by centrifugation then applied to arrays, which were incubated for 16 hours at 40°C at 60 rpm. Arrays were stained with SAPE labeling mix (1x MES staining buffer, 2 mg/ml BSA, 10 μg/ml streptavidin-phycoerythrin), and washed on an Affymetrix fluidics station, then scanned. (see PMID: 21548937)
Scan protocol
As described in PMID: 21548937
Data processing
Feature signal was extracted from the GMAP arrays using Affymetrix Power Tools (APT, http://www.affymetrix.com webcite). The GMAP chip contains triplicate features per shRNA, which we summarized by using the median value. GC-background (GCbg) correction for non-specific probe binding was performed with APT, using feature signals derived from 33,894 GC-background probes on the array. Normalization of replicate arrays was performed with the Bioconductor affy package in R, using Cyclic Loess based on the 'MA-plot' of pairs of arrays. As a further normalization step, all T0 samples were normalized using Quantile Normalization. This normalization was also applied to all T1 and (separately) T2 samples. The log2 measurement values are present in the processed matrix.
