Primary antibody oligonucleotide conjugation

For the species mixing experiment, anti-human CD29 and anti-mouse CD29 antibodies were purchased from Biolegend (cat. 303021, 102235) and conjugated per antibody using a ThunderLink kit (Expedeon cat. 425–0000) to distinct 20 bp 3’ amine-modified HPLC-purified oligonucleotides (IDT) to serve as hybridization handles. Antibodies were conjugated at a ratio of 1 antibody to 3 oligonucleotides (oligos). In parallel, oligos similar to current antibody sequencing tags were directly conjugated at the same ratio for comparison. Sequences for the hybridization oligonucleotides and directly conjugated oligos were designed to be compatible with the 10x feature barcoding system by introducing a reverse complementary sequence to the bead capture sequence, alongside a pool and antibody specific barcode for resolution. Conjugates were quantified using Protein Qubit (Fisher cat. {"type":"entrez-protein","attrs":{"text":"Q33211","term_id":"75281052","term_text":"Q33211"}}Q33211) for antibody titration and flow validation. In addition, we orthogonally quantified the antibodies using protein BCA (Fisher cat. 23225). For the human donor mixing experiment, CD4 and CD20 antibodies (Biolegend cat. 300541, 302343) were conjugated as described above. Antibodies used in PBMC and comodality experiments are listed in Supplementary Table 1 and 2.