Supplementary Materials and Methods
Genome-wide pooled shRNA Screen
Genome-wide pooled short hairpin RNA (shRNA) screen to identify genes
essential for p53-mediated apoptosis in MCF-7 cells treated with RITA was
performed using a lentivirus-delivered pool of 27,290 shRNAs targeting 5,046 known
human genes.
Experimental procedure
Six million cells were infected with shRNA library at MOI of 50% (this will
allow each individual shRNA of the library with the complexity of about 27,000 to be
delivered to at least 100 cells). After 2 days infected cells were selected using
Puromycin for 3 days. Then cells were allowed to proliferate for one week. After that
cells were split and treated with 1 μM of RITA or DMSO for 24 hours, re-plated and
allowed to propagate for 4 days, after which cells were collected for the analysis. This
treatment allows death of a part of tumor cells (70 %) with further expansion of those
which survived. Comparison of populations of survived cells versus untreated cells
allows to identify in one screen those shRNA which confer sensitivity to RITA (lost in
the process of selection, synthetic lethal nodes) and those which confer resistance to
RITA (enriched in the process of screening, resistance nodes). Each condition was
performed in triplicate. The relative enrichment or loss of individual bar codes were
detected by Illumina sequencing.
Data processing
1) For each sample, total number of reads were normalized to 20x106.
2) Twenty additional reads were added to each shRNA to minimize false
positives in the low-abundance tail of the shRNA library distribution, where loss of a
significant fraction of cells can happen randomly.
3) For both non-treated and RITA-treated samples, arithmetic mean was
calculated for each shRNA targeting Luciferase. The ratio between treated cells versus
untreated was calculated. The average ratio in shLuc MCF-7 cells is 1.09, which
presents the ratio upon RITA treatment in cells without knockdown of any gene.
4) For each sample, all reads were normalized with the arithmetic mean of
shLuc.
5) Then arithmetic mean was calculated for each shRNA in both non-treated and
RITA-treated samples. The relative ratio between treated cells versus untreated was
calculated.
6) shRNA whose abundance was significantly different between control and
RITA treatment were selected based on three criteria most commonly used by
biologists in analysis of high-throughput shRNA screens: (i) p value, (ii) FDR, and (iii)
p values of the weighted Z scores, p(wZ)). The p(wZ) values integrate the information
from multiple shRNAs targeting a single gene, thus showing the similarity of the
effect of these multiple shRNAs and minimizing the impact of possible off-target
effects.
7) Hierarchical analysis demonstrated the efficient clustering of the DMSO and
RITA biological replicates in MCF-7 cells and predicted two groups of genes
according to above criteria: synthetic lethal nodes, depletion of which sensitized cells
to RITA treatment (SLNs; the ratio of read counts between DMSO and RITA > 1) and
resistance nodes, depletion of which rendering cells resistant to RITA treatment (RNs;
the ratio of read counts between DMSO and RITA < 1).
8) Validation of genome-wide shRNA screen. 15 genes were selected from
screen randomly and re-tested for validation purpose, using independen shRNAs from
Sigma, 4-5 per gene. Results were compared with results of statistic analysis of screen
data under three different levels of strengths. A cut-off based on three criteria (i) p
value < 0.05, (ii) FDR < 0.1, and (iii) p(wZ) < 0.1 was selected in present study,
which is a highly stringent cut-off producing false negatives (Table S2). Nevertheless,
this cut-off was not relaxed, since lower criteria not only decreased the false negatives
but also increased false positives. In addition, we obtained similar results of pathway
analysis for both stringent and relaxed criteria.