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  • br Integrated raw data and

    2022-05-18


    Integrated raw data and preprocessed data
    This paper
    Experimental Models: Cell Lines
    A gift from Dr. Lukas Sommer
    Recombinant DNA
    The human kinase library plasmid kit Addgene Kit# 1000000014
    ORFeome Human Entry Collection Phosphatase Dharmacon Cat# OHS4941
    pDEST pcDNA5 FRT TO-eGFP A gift from Dr. Anne-Claude Gingras N/A
    pDEST 30 Triple Flag pcDNA5 FRT TO A gift from Dr. Anne-Claude Gingras N/A
    Addgene
    Software and Algorithms
    Cytobank Cytobank https://www.cytobank.org/
    Concatenation tool Cytobank https://support.cytobank.org/hc/en-us/articles/
    Normalizer Finck et al., 2013 https://github.com/nolanlab/bead-
    normalization/releases
    Single cell debarcoder Zunder et al., 2015 https://github.com/nolanlab/single-cell-
    BP-R2 analysis
    debarcoder
    Lun et al., 2017 https://github.com/BodenmillerGroup/Adnet
    t-SNE van der Maaten and Hinton, 2008 https://github.com/jkrijthe/Rtsne
    STRING Szklarczyk et al., 2017 https://string-db.org/
    Shape-based clustering Genolini et al., 2015 R package ‘kml’
    CONTACT FOR REAGENT AND RESOURCE SHARING
    Further information and requests for resources and reagents should be directed to and will be fulfilled by Caffeine Lead Contact, Bernd Bodenmiller ([email protected]).
    EXPERIMENTAL MODEL AND SUBJECT DETAILS
    METHOD DETAILS
    Cloning
    The human kinase library plasmid kit, containing open reading frames (ORFs) in Gateway Entry vectors, was provided by William Hahn and David Root (Johannessen et al., 2010; Yang et al., 2011) via Addgene (Kit # 1000000014). The human phosphatase library was obtained from Dharmacon (OHS4941, ORFeome Human Entry Collection Phosphatase). Destination vectors, including pDEST pcDNA5 FRT TO-eGFP and pDEST 30 Triple Flag pcDNA5 FRT TO, were kindly provided by Dr. Anne-Claude Gingras at Mount Sinai Hospital, Toronto, Canada (Couzens et al., 2013). Expression vectors encoding the FLAG- or GFP-tagged fusion proteins were generated via Gateway Cloning and sequenced before transfection. Vectors for kinase-dead mutants, including pFLAG-CMV-hErk1 (K71R) (Addgene plasmid # 49329), pCIG AKT3 (K177M) (Addgene plasmid # 73051), pMCL-HA-MAPKK1-8E (K97M) (Addg-ene plasmid # 40811), IRES-GFP-AXL-KD (K567R) (Addgene plasmid # 65498), and FLAG.PKCepsilon.K/W (K437W) (Addgene plasmid # 10796) were gifts from Melanie Cobb, Joseph Gleeson, Natalie Ahn, Aaron Meyer, and Alex Toker, respectively (Baek et al., 2015; Cenni et al., 2002; Meyer et al., 2015; O’Neil et al., 1990).
    HEK293T cell transfection and stimulation
    HEK293T cells were seeded at the density of 0.7 million per well in 6-well plates. After 24 h, cells were transfected with 2 mg plasmid and 4 mL of jetPRIME (PolyPlus) per well with the standard protocol provided by the manufacturer. For kinase and phosphatase dou-ble transfection experiments, cells were transfected with plasmids encoding GFP-tagged kinases and FLAG-tagged phosphatases 16 h and 24 h after seeding, respectively. Half the amounts of plasmid and jetPRIME were used in each round for co-overexpression experiments. At 18 h after transfection, EGF (Peprotech) was added to a final concentration of 100 ng/ml. At 20 min before a given EGF stimulation time point, 5-iodo-deoxycytidine (IdU) was added to the medium at the final concentration of 10 mM. At 2 min before a given EGF stimulation time point, medium was replaced by 1X TrypLE to induce cell detachment. At the time point, paraformaldehyde (PFA, from Electron Microscopy Sciences) was added to the cell suspension to a final percentage of 1.6%, and cells were incubated at room temperature for 10 min. If EGF stimulation was not necessary in the experiment, cells were directly harvested and crosslinked with PFA.