a, Lineage diversity of PRISM cell lines. The 489+
cancer cell lines tested span more than 23 tumor types. Lineages with fewer
than 10 cell lines are listed on the right. b, Experimental
protocol. Cell lines are grouped by doubling time into pools of
approximately 25 cell lines. One pool is plated onto each assay plate.
Compounds are transferred by pin transfer from a source compound plate (HTS
and HTS002 screens), or cells are plated directly onto assay-ready plates
generated by acoustic dispensing of compounds (MTS004, MTS005, and MTS006
screens). In either case, compound plates are shared by all replicates of
each treatment condition. After incubation and lysis, all assay plates
generated by a given compound plate are grouped and collapsed into 3
(HTS002, MTS005, and MTS006 screens) or 6 (HTS, MTS004 screens) detection
plates so that each detection plate receives 1 or zero copies of each pool.
Ten control barcodes are then spiked into each detection plate well (HTS002,
MTS005, and MTS006 screens). Detection plates are amplified by PCR and
detected using Luminex FLEXMAP 3D instruments. c, Data
processing workflow. Median Fluorescence Intensity (MFI) values are
calculated from fluorescence values for each replicate-condition-cell line
combination and are log2-transformed. Assay plates wells are normalized,
median-collapsed, and compared to the normalized medians of other assay
plate wells in the same well position that have been dosed by the same
compound plate. A robust z-score is calculated, and assay plate wells with a
|z-score| > 5 are filtered. Strictly standardized mean differences
(SSMD) are calculated between positive and negative control conditions for
each cell line on each assay plate. Cell line-assay plate combinations with
SSMD < 2 are filtered by a control-separation filter to generate the
log MFI data matrix. In datasets with control barcodes added, data are
normalized with respect to the median of control barcodes to generate the
MFI normalized data matrix. Data are DMSO-normalized and pooling artifacts
are corrected using ComBat to generate the log fold-change data matrix. Up
to 3 independently treated plates (range 1–3 based QC filtering) in
one screen are median-collapsed to generate the collapsed log fold-change
data matrix.