Extended Data Fig. 1: Design features of the UltraSelex RNA libraries.

a, Conventional SELEX scheme. The input dsDNA pool (round 0) is in vitro transcribed into RNA to yield the initial RNA pool, which is incubated with the immobilized target. After removal of unbound RNA by washing, the bound fraction is eluted, reverse-transcribed into cDNA and PCR-amplified. The obtained enriched dsDNA pool is used as input for the next iteration, and this cycle is repeated until binders dominate. Binders are then identified, based on their abundance, by sequencing. b, Design of the partially structured RNA pool used in all UltraSelex experiments reported here, and stepwise processing to yield the barcoded sequencing library. The RNA pool contained two constant primer-binding regions (purple and blue)¹⁷, two randomized stretches of 26 nt each (N26) and a constant 12 nt internal hairpin (H12) thought to increase the abundance of high-affinity aptamers in the starting pool¹⁶. An aliquot of the same RNA pool was used previously for the isolation of SiR-binding aptamers by conventional SELEX¹⁸. The offset PCR system was designed to reduce bias in the sequencing of templates with variable and constant regions, thereby achieving balanced per-base nucleotide compositions⁴⁶. Equimolar amounts of seven forward primers (Frw) and seven reverse primers (Rev) containing 0–6 inserted offset nucleotides (golden) were mixed and used for PCR amplification, generating length-heterogenous sequencing libraries. After offset PCR with the primer mixture, the Illumina primers, indices and barcodes were attached in another PCR reaction (barcode PCR). Before sequencing, a custom balancer oligonucleotide was added. For sequences, see Supplementary Table 2. c, Detailed architecture of the complete sequencing template.