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  • N K L designed and performed the CRISPR

    2019-10-14

    N.K.L. designed and performed the CRISPR screen and validation experi-ments, isolated tumor Nutlin-3 for ChIP-seq, prepared samples for RNA-seq, per-formed all functional experiments related to RORg inhibition in vitro and in vivo, and performed histologic analysis and live imaging experiments. L.P.F. carried out CRISPR screen validation and related bioinformatic analysis, in vivo RORg target analysis, protein expression, cytokine analysis, RNA-seq library prepa-ration and helped with manuscript and Þgure preparation. N. Rajbhandari analyzed Rorc / KPf/fC mice and helped with in vivo drug studies and CRISPR screen validation; R.F., T.G., and L.A.E. provided experimental help; P.S., N.E.L., M.H., K.M.F., R.S., and S.B.R. performed bioinformatics analysis related to RNA-seq and CRISPR screen; P.N. and H.H. carried out the single-cell RNA-seq and W.L. and M.H. performed related computational analysis; A.D. and A.J.D. performed ChIP-seq; K.G., N. Robertson, and P.D.A. performed all ChIP-seq analysis; and for tissue microarray (TMA) anal-ysis, C.M.S. and M.W. analyzed and interpreted TMA staining, C.M.S. created Þgures and wrote the report, I.Z. performed statistical analysis, M.E. created Scorenado and provided technical advice, J.A.G. performed TMA immunohis-tochemistry (IHC) staining, and E.K. created the TMAs and collected clinical data. T.I., D.V.H., A.M.L., and P.D.A. provided experimental and/or computa-tional advice and comments on the manuscript. N.K.L., L.P.F., and M.K. helped write the paper. T.R. conceived of the project, planned and guided the research, and wrote the paper.
    DECLARATION OF INTERESTS
    This work includes data Þled as a provisional application SD2019-243. T.R. is a consultant for Orphagen Pharmaceuticals, and D.V.H. serves on the ScientiÞc Advisory Board of Five Prime and is a consultant for Celgene.
    REFERENCES
    Cook, D.N., Kang, H.S., and Jetten, A.M. (2015). Retinoic acid-related orphan receptors (RORs): regulatory functions in immunity, development, circadian rhythm, and metabolism. Nucl. Receptor Res. 2, 101185.
    Hu, Y., and Smyth, G.K. (2009). ELDA: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays. J. Immunol. Methods 347, 70Ð78.
    Martin, M. (2011). Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17, 10Ð12.
    Van den Broeck, A., Vankelecom, H., Van Delm, W., Gremeaux, L., Wouters, J., Allemeersch, J., Govaere, O., Roskams, T., and Topal, B. (2013). Human pancreatic cancer contains a side population expressing cancer stem cell-associated and prognostic genes. PLoS ONE 8, e73968.
    van Laarhoven, T., and Marchiori, E. (2012). Robust community detection methods with resolution parameter for complex detection in protein protein interaction networks. In Pattern Recognition in Bioinformatics, T. Shibuya, H. Kashima, J. Sese, and S. Ahmad, eds. (Springer).
    Wartenberg, M., Cibin, S., Zlobec, I., Vassella, E., Eppenberger-Castori, S., Terracciano, L., Eichmann, M.D., Worni, M., Gloor, B., Perren, A., and Karami-topoulou, E. (2018). Integrated genomic and immunophenotypic classiÞcation of pancreatic cancer reveals three distinct subtypes with prognostic/predictive signiÞcance. Clin. Cancer Res. 24, 4444Ð4454.
    STAR+METHODS
    KEY RESOURCES TABLE
    REAGENT or RESOURCE
    SOURCE
    IDENTIFIER
    Antibodies
    chicken anti-GFP
    Abcam
    Rabbit anti-RORg
    Thermo Fisher
    Mouse anti-E-Cadherin
    Anti Mouse Pan-Keratin
    Anti Celsr1
    Anti Celsr2
    Anti-Hmga2
    Anti-mouse EpCAM-APC
    Anti mouse CD31-PE
    Anti-mouse Gr-1-FITC
    Anti-mouse CD11b-APC
    Anti-mouse CD4-FITC
    Anti mouse CD4-PaciÞc blue
    Anti-mouse CD8-PE
    Anti-mouse IL-17-APC
    Anti-mouse PDGFRa-BV421