Oligonucleotide conjugation kit

Antibody–adapter conjugates were generated by random amino-conjugation between 100 µg of antibody purified in PBS in the absence of glycerol, BSA and sodium azide and 5′ aminated, barcode-containing oligonucleotides (Integrated DNA Technologies) using the Oligonucleotide Conjugation Kit (Abcam) according to the manufacturer’s protocols. Before conjugation, 200 µM adapter oligos resuspended in 1× PBS were annealed to an equimolar amount of 200 µM Tn5MErev (5′-[phos]CTGTCTCTTATACACATCT-3′) in 1× PBS to yield 100 µM annealed adapters. In all cases, primary antibodies were conjugated with an estimated 10:1 molar excess of adapter to conjugate. The sequences of adapters used are listed in Supplementary Table 1.

The oligonucleotide containing the Zika virus target sequence (bold) (ZTargetamine: 5’-amine-C6-MMT-TAAAGATGGCTGTTGGTATGGAATGGAGATAAGGCCCAGGAAAG-3’) was covalently attached to an IgG secondary antibody (goat anti-rabbit IgG, whole molecule; Sigma), using an oligonucleotide-conjugation kit according to the manufacturers’ procedure (Abcam, Cambridge, MA, USA). HeLa cells were grown in Falcon 8-well cell culture slides, fixed with 10% formalin/PBS, permeabilized with saponin (0.05%) and incubated with primary (anti-Akt1/2/3 (Santa Cruz, Dallas, TX, USA)) and oligonucleotide-labeled secondary antibody using standard protocols as for immunofluorescence staining, and TN-RCA performed in situ essentially as described above, washed with PBS, and photographed with a fluorescent microscope (BZ-X710, Keyence, Itasca, IL, USA).