Rabbit immunoglobulins

Chromatin immunoprecipitation was performed as described previously (McNee et al., 2017 (link)). Briefly, 48 h post transfection with siRNA, double-strand breaks were induced in U-2-OS-FokI cells by addition of 1 μM Shield1 ligand (Clontech) and 1 μM 4-hydroxytamoxifen (Sigma-Aldrich) for 4 h, or left untreated. Samples were crosslinked with 1 % formaldehyde and neutralized with 0.125 M glycine. Cells were lysed and DNA sheared to 300-1000 bp by sonication. Samples were pre-cleared with rabbit immunoglobulins (Dako) and chromatin was co-immunoprecipitated with antibodies to BOD1L, SETD1A, RIF1, histone H3, H3K4me1, H3K4me2 and H3K4me3. Protein-DNA complexes were washed and eluted from magnetic protein G beads, cross-links were reversed and samples treated with proteinase K. DNA was purified using a PCR purification kit (Qiagen) and quantified by qPCR using 4 primer pairs (see Figure 6O for location of amplicons, and key resources table for sequences).

Hearts were embedded in OCT (Optimal Cutting Temperature; Tissue-Tek, Zoeterwoulde, The Netherlands) and sections of 10 μm thickness were collected. For macrophage and T cell content, subvalvular plaques were stained with MOMA-2 antibody (BMA Biomedicals, Switzerland) and anti-CD3 (Dako A0452) respectively, using rabbit immunoglobulins (Dako, Solna, Sweden) as negative controls. DAB detection kit was used for color development (Vector Laboratories, CA) and the sections were counterstained in haematoxylin. For assessment of the collagen content of the plaques, subvalvular sections were stained with Van Gieson Solution Acid Fuchsin (Sigma-Aldrich). To assess the necrotic core, sections were stained with haematoxylin/eosin and the area was determined as the acellular area, lacking nuclei and cytoplasm, under the fibrous cap of lesions. All stainings were quantified with Image-Pro-Plus 4.5 software (Media Cybernetics).