Crosslinking RaPID screening (XL-RaPID)

RaPID screens were adapted from protocols previously described³⁰. In brief, puromycin-ligated randomised mRNA libraries (NNK6–NNK12) were in vitro translated (30 min, 37 °C then 12 min, 25 °C) using a custom transcription/translation mixture supplemented with ClAc-D-Tyr-tRNA^fMet_CAU (25 µM) and pBpa-tRNA^Asn_CAU (25 µM). Methionine and 10-formyl-5,6,7,8-tetrahydrofolic acid were not included in the translations. First-round translations were performed on a 150-µL scale, and subsequent rounds on a 10-µL scale. Following the addition of 200 mM EDTA (pH 8.0), the translated mixture was reverse transcribed with M-MLV RTase, RNase H minus. The resulting mixture was first buffer exchanged into selection buffer (50 mM HEPES, 150 mM NaCl, 2 mM DTT, 0.1% Tween, pH 7.5) using a 1 mL sephadex column (G-25 fine, GE Healthcare) before the addition of 2× blocking buffer (50 mM HEPES, 150 mM NaCl, 2 mM DTT, 0.1% Tween, 0.2% (w/v) acetylated bovine serum albumin, pH 7.5). Biotinylated BRD3-BD2 (200 nM) was then added, and the resulting mixture was irradiated at 365 nm in a Longwave Ultraviolet Crosslinker (model CL-1000 L, UVP, analytikjena) for 30 min at 0 °C. The irradiated mixture was then incubated with Dynabeads M-280 streptavidin (Life Technologies) for 15 min at 0 °C. The bead-immobilised protein was washed with 5 M guanidine HCl (2 × 20 min) at 0 °C before further washing with ice-cold selection buffer (3 × 1 min). PCR solution was added and the retained peptide-mRNA/DNA hybrids were eluted from the beads by heating (95 °C, 5 min). Library enrichment was assessed by quantitative real-time PCR relative to the input DNA library using primers T7g10M.F46 and CGS3an13.R22. The recovered DNA was amplified by PCR and used as the input for the next selection round.

Following five rounds of crosslinking RaPID selection, double-indexed libraries (Nextera XT indices) were prepared from recovered library DNA from rounds one to five and sequenced on the Illumina HiSeq 4000 or NovaSeq platform with single-ended 100 bp reads. Each DNA sequence was converted to a peptide sequence and ranked by total read number. For primers used to generate reagents and prepare the library for sequencing, see Supplementary Table S1.