Bcivi

To assay the extent of angelicin modification, we selected the restriction endonuclease BciVI as its recognition sequence harbors a central ‘AT’ motif that becomes unrecognizable in the presence of an angelicin modification. Plasmid vector pBluescript SK+ (https://www.addgene.org/vector-database/1951/) was modified with 0, 100, and 500 uM angelicin prior to digestion with BciVI (NEB). 2.5 micrograms of DNA modified at each angelicin concentration were cut with either 4 or 8 units of restriction enzyme. Digests were analyzed on a 4150 TapeStation (Agilent) using a D5000 Screentape.

On-chip synthesized oligonucleotides were purchased from Cutsom Array, Inc. A total of 3692 ATGL primers were generated for each of the two libraries. The oligonucleotides (104 bp length: 60 bp primer + 44 bp adapter sequences) were amplified via PCR and adapter sequences were removed using restriction enzymes BciVI (New England BioLabs GmbH, R0596S) and BspQI (New England BioLabs GmbH, R0712S). Purified via an 8% acrylamide 8 M urea gel. Bands of 60 bp size were cut out and gel pieces were incubated in 200 µl dH2O at 4 °C for 24 h to resolve the 60 bp oligonucleotides.
Deep mutagenesis protocol from E. Wrenbeck et al. was used^{49 (link)}. Minor changes were applied to increase the mutational power and reduce the WT background (Plasmid Safe digest and DPNI digest).