High capacity neutravidin agarose resin

The SELEX protocol was adapted from a reported method.^{28 (link)} A schematic of the SELEX procedure is shown in Fig. 1, and the conditions used in the individual rounds are summarized in Supplementary Table 2. Broadly, positive selection was conducted for Round 1, in which 4 × 10⁷ thawed mixed T cells, depleted of dead cells (Miltenyi), were incubated with 40 nmol ssDNA library (~10¹⁶ individual sequences) for 1 h at 4 °C in binding buffer. Bound aptamers were extracted and amplified by PCR using Phusion High Fidelity DNA Polymerize (NEB) with forward and reverse primers. Strand separation was performed with High Capacity Neutravidin Agarose Resin (Thermo Scientific), as described previously,^{28 (link)} and the FAM-labeled ssDNA aptamer pool was used in next round. For Rounds 2–5, the ssDNA aptamer pools were incubated with thawed PBMCs depleted of dead cells, a process termed competitive selection. After three washes, T cells and bound ssDNA sequences were then enriched using a Pan T Cell Isolation Kit (Miltenyi). The ssDNA pool was then extracted and incubated with 10⁷ CD3⁻CD8⁻ J.RT3-T3.5 cells at 4 °C as a form of negative selection in each round, and unbound ssDNA sequences were PCR amplified and used to generate ssDNA aptamer pools for use in the sequential round. The wash and binding buffer formulations, as well as folding conditions, are as described previously.^{28 (link)}