External ID: CBX7-Alpha-primary

Protocol: The protocol for the CBX7 AlphaScreen that we use is as follows: We create a standard alpha buffer (50mM HEPES, 150mM NaCl, 0.1% w/v BSA, 0.01% w/v Tween20) at pH 8.0 and make up two stock solutions at 2x final concentration in the alpha buffer. Solution A contains Human His6-CBX7 is used with biotinylated-H3K27me3 (residues 20-34), at 100nM and 50nM respectively. Solution B contains 20nM streptavidin donor beads and 20nM nickel acceptor beads. For 384 well assay formats, 10uL of solution A is added to each well of the assay plate and the plate is spun at 1000 rpm for 30 seconds. 100 nL of experimental ccompounds from stock plates are delivered by robotic pin transfer using a Janus Workstation (PerkinElmer), allowing the compounds to interact with CBX7 binding prior to assay measurement, followed by another spin and an incubation room temperature. Finally, 10uL of solution B is added to each well, the plate is spun again and then incubated at room temperature. AlphaScreen measurements are performed on an Envision 2104 (PerkinElmer) utilizing the manufacturer's protocol that has the correct excitation and emission wavelengths, cutoff filters, delay time, etc. We also do a crosstalk calculation through the Envision software to correct for luminescence for adjacent wells while reading the plate. The signal is then normalized to DMSO control wells on the compound plate prior to creation of the IC50 curves.

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External ID: RMI-FANCM-MM2

Protocol: FP HTS. Screening took place at the University of Wisconsin Small Molecule Screening and Synthesis Facility. A master mix of RMI core complex and F-MM2 (30 microL per well) was plated in black 384 shallow well plates (ThermoFisher, Waltham, MA), using a BioTek "MicroFlo Select" reagent dispenser. Compounds were added using a Beckman FX liquid handler; 0.33 microL of 10 mM stock was added for a final compound concentration of 33 microM. MM2 and cMM2 were each added to 4 wells of master mix per plate to a final concentration of 10 muM to serve as controls. Following compound addition, plates were covered, centrifuged briefly, and incubated for 20 minutes at room temperature. FP measurements were taking using a Tecan "Safire 2" microplate reader. Instrument settings were as follows: top read, EX 470, EM 525/20, G-factor 0.89947. A suitable gain was calculated from the first plate of each day. Z' scores for were calculated for each plate; plates with Z' scores <0.5 were rerun prior to analysis.

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