Ethylene glycol bis

Arabidopsis accession Columbia (Col-0) and myc2 were grown at 22 °C under a 16 h light/8 h dark photoperiod. The aerial part of two-week-old seedlings were dual cross-linked with 1.5 mM ethylene glycol bis (succinimidylsuccinate) (Thermo Fisher Scientific, 21,565) for 30 min and 1% formaldehyde (SIGMA, F8775) for 10 min, and then quenched with 0.2 M glycine for 5 min at room temperature. Harvested samples were stored at -80 °C for ChIA-PET assay. For ChIP-seq assay, samples were crosslinked with 1% formaldehyde for 10 min, quenched with 0.2 M glycine at room temperature, and then stored at -80 °C until use. Samples were immediately frozen in liquid nitrogen and stored at -80 °C for RNA-seq assay.

Whole-cell lysate (WCL) or nuclear fraction was prepared from 3T3-L1 preadipocytes transduced with SETD5-V5 before differentiation induction. For detection of SETD5 interaction with CDC20, preadipocytes were induced with MDI for 9 h and then treated with 10 μM MG132 for 12 h before harvesting. WCL or nuclear fraction was subjected to immunoprecipitation using anti-V5 antibody (Thermo Fisher Scientific, R960-25, 2 μg) or control mouse IgG. Immunoprecipitates were eluted with SDS sample buffer and subjected to immunoblot analysis using indicated antibodies. For detection of SETD5 interaction with C/EBPβ, 3T3-L1 preadipocytes transduced with SETD5-V5 were cross-linked with 1.5 mM ethylene glycol bis(succinimidylsuccinate) (Thermo Fisher Scientific) for 30 min at followed by second cross-linking by addition of 1% formaldehyde for 10 min. After cross-linking, nuclear fraction was prepared and subjected to immunoprecipitation with Dynabeads Protein G (Thermo Fisher Scientific) conjugated with anti-C/EBPβ (Santa Cruz Biotechnologies, sc-150×, 6 μg) or control rabbit IgG. Immunoprecipitates were eluted with SDS sample buffer, de-crosslinked at 37 °C for 3 h, and subjected to immunoblot analysis.