0.5 to 1.0 µM PRC2 complex with its accessory subunits was cross-linked with 5-20 µg bis[sulfosuccinimidyl] suberate (BS3) (ThermoFisher #21585) in 20 mM HEPES pH 7.5 (at 25°C), 150 mM NaCl, 1 mM TCEP, in a total reaction volume of 85 µL. Reactions were allowed to proceed for 30 min at 25°C before Tris-HCl pH 8.0 (at 25°C) was added to a final concentration of 30 mM, to quench the reaction, for 15 min at 25 °C. Crosslinking efficiency was assessed by subjecting a portion of the product to 10% SDS-PAGE. The crosslinked product solutions were adjusted to pH 8.0 by adding 20 µL of 1 M Tris-HCl pH 8.0 (at 25 °C), reduced in 10 mM TCEP for 30 min at 60 °C and then alkylated in 40 mM 2-chloroacetamide for 20 min at 25 °C in the dark. Mixtures were then digested with 1:100 trypsin:protein mass ratio overnight at 37 °C with shaking on a Thermomixer (Eppendorf). Protein digestions were stopped by acidification through adding formic acid to 1 % (v/v). Digested peptides were purified using OMIX C18 Mini-Bed pipette tips according to the manufacturer’s instructions, dried in a vacuum concentrator and reconstituted into 20 µL of 0.1 % formic acid prior to mass spectrometric analysis.
The peptides were analysed by LC-MS/MS on an Orbitrap Fusion Tribrid Instrument connected to an UltiMate 3000 UHPLC liquid chromatography system (Thermo-Fisher Scientific) as described above for RBDmap.
pLink and pLink2 was used to identify BS3-crosslinked peptides with a false discovery rate of 0.05. In addition, the crosslinked peptides were kept for downstream structural analysis only if they have been identified in at least two independent replicates or if they have been identified with a p-value of less than 10-5. 2D representations of crosslinked peptides and proteins were generated using xiNET. R scripts used for data retention and analysis downstream of pLink can be downloaded from GitHub: https://github.com/egmg726/crisscrosslinker.