Oligonucleotide to antibody conjugation

Anchor oligonucleotides (Supplementary Table ¹) were conjugated to secondary antibodies for immunolabeling of cardiac samples. Lyophilized oligonucleotides were resuspended in PBS (pH 7.4) to 100 µM and kept at −20 °C for long term storage until required for conjugation. AffiniPure Goat Anti-Mouse secondary antibodies (affinity purified, #115-005-003, Jackson ImmunoResearch, PA) were conjugated using click-chemistry as described by Schnitzbauer et al.² Briefly, the antibody was incubated with 10-fold molar excess DBCO-sulfo-NHS-ester (Jenabioscience, Germany) for 45 min. The reaction was quenched with 80 mM Tris-HCl (pH 8.0) for 10 min and then desalted using 7 K MWCO Zeba desalting columns (Thermo Fisher). A 10-fold molar excess of the azide modified oligonucleotide was then incubated with the DBCO-antibody mixture overnight at 4 °C. Subsequently the antibody was purified using 100 K Amicon spin columns (Sigma). The absorbance of the oligonucleotide-conjugated fluorophores (Cy3 or Cy5) was recorded with a Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham) and used to quantify the degree of labeling for each conjugation, typically achieving >1–3 oligonucleotides per antibody.