GoKawiilSource and culture conditions of NextGen cancer models
The source of each of the NextGen models is provided in Supplementary Table 1. In brief, breast cancer organoid models were derived by the laboratories of A.V.L., S.O. and D.L.S. Most of the NextGen CNS tumour models were derived by the laboratory of K.L.L. Colorectal and oesophagus–stomach cancer organoid models were derived by staff at the Cancer Cell Line Factory at the Broad Institute. Ovarian and uterus cancer organoid models were derived by the laboratory of S.J.H. Pancreas cancer organoid models were derived by the laboratories of A.J.A. and S.R. Prostate cancer organoid models were derived by the laboratory of H.B. and the laboratory of Y. Chen. Additional models derived by the HCMI were obtained via the American Type Culture Collection (ATCC). Models were confirmed for the absence of mycoplasma contamination (Mycoplasma PCR Detection kit; ABM, G238) and authenticated by short tandem repeat (STR) profiling (LabCorp) before expansion. These procedures were repeated monthly during expansion and screening.
Organoid media were prepared using the recommendations provided for each of the models (Supplementary Table 1). The conditioned media were prepared by culturing WNT3A-producing L cells (L Wnt-3A; ATCC, CRL-2647) and R-spondin-1-producing 293T cells (Cultrex R-spondin 1 (RSPO1) cells; Trevigen, 3710-001-K) in DMEM with 10% serum. After reaching 80% confluency, cells were washed once with PBS and incubated with Advanced DMEM–F12 medium (Thermo Fisher, 12634028) containing 100 mM HEPES, 1% penicillin–streptomycin and 1% 100× Glutamax (Ad+++) for another week. The conditioned medium from this culture was then collected, filtered and stored at –80 °C before being used for organoid cultures. All organoids were grown initially as domes composed of 75% growth-factor-reduced, Phenol Red-free Matrigel (50 μl per dome; Corning, 356231) in the recommended medium diluted 1:1 with Ad+++. The screening of organoids was conducted either in Matrigel dome cultures or in adherent cultures on Matrigel-coated plates (see the section ‘Genome-wide CRISPR screening of organoids’ below).
The NextGen CNS models were grown in tumour stem medium, the composition of which is described in Supplementary Table 1. The conditioned medium was collected routinely during expansion and preserved for subsequent cultures. Cells were seeded in a mixture of 30% conditioned medium and 70% fresh medium. Some of the models were tested for their ability to grow on a Matrigel-coated plate. In brief, cells were plated in 1% Matrigel-coated flasks and cells attached to the coating were carried forward as adherent models. Models that failed to attach to coated plates were grown as spheroids in ultra-low attachment (ULA) vessels (Corning, 3814). Models that were initially derived as adherent cultures on laminin-coated plates were maintained in the same conditions. To coat plates, Matrigel or laminin (Corning, 354232) was diluted in ice-cold PBS at a concentration of 1%. This solution was then added to culture vessels at approximately one-half the recommended volume of medium for a given vessel (for example, 5 ml solution in a T75 flask). Flasks were incubated at 37 °C overnight, and the solution was aspirated immediately before plating the cells. The growth format of each model is listed in Supplementary Table 1.
Preparation of samples for WGS and RNA-seq
To generate pellets from the organoids, the medium was first aspirated and then the Matrigel domes were scraped and transferred to a 20× volume of pre-warmed TrypLE (Thermo Fisher, 12604021) with 10 µM ROCK inhibitor (Peprotech, 129830-38-2). This mixture was incubated at 37 °C for 30 min. Subsequently, the cells were pelleted by centrifugation, resuspended in a mixture of medium and Matrigel and seeded at a density of 50,000 cells per dome. After 1 week of growth, cell pellets were collected without ROCK inhibitor for WGS and RNA-seq.
For the NextGen CNS tumour models, cells were seeded and propagated for 7–14 days, depending on their growth, and dissociated with TrypLE for 5 min at 37 °C. Following dissociation, the cells were counted, and the pellets of 1 × 106 cells were collected and submitted for WGS and RNA-seq.
WGS analysis
WGS for all the DepMap NextGen models was conducted at the Broad Institute Genomics Platform using the PCR-free human WGS procedure. In brief, libraries were constructed and sequenced at 30× coverage on either an Illumina NovaSeq 6000 or NovaSeq X system, with the use of 150 base-pair paired-end reads. The FASTQ files from sequencing were de-multiplexed, aggregated and aligned using the DRAGEN germline pipeline to create BAM files.
Mutation calling from WGS
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