Genotyping
We used error-prone PCR61 to prepare a library of avGFP random mutants. Random mutagenesis was performed using an in house analogue of the Diversity PCR Random Mutagenesis kit (Clontech, 50x mix: 0.2 мМ dGTP, 0.2 мМ dATP, 1 мМ dCTP, 1 мМ dTTP)). We have optimized the conditions of the PCR by varying the buffer composition and the number of cycles to obtain on average 4 mutations per coding region. One of the primers used for PCR contained a stretch of 20 random nucleotides, which introduced the molecular barcode downstream to the GFP coding sequence. The library was digested with NheI and NotI restriction enzymes and subcloned into the construct mKate2-rigid_linker-EGFP /pQE-30 instead of the EGFP gene. The final library diversity was estimated to be about 300,000 clones. The C-terminal half of the gene linked to the barcodes was obtained by digesting an aliquot of the plasmid library with SpeI and NotI. To link the N-terminal half of the gene with the corresponding barcodes of the C-terminal we digested the plasmid library with SpeI and AvrII, purified it through a gel, eliminating the short fragment corresponding to the C-terminal half of the gene, and then self-ligated it. Self-ligation was performed at low DNA concentration in the presence of both SpeI and AvrII to avoid intermolecular ligation. The resulting library was transformed into E. coli, grown on Petri dishes and purified again. The diversity of the resulting library of self-ligated plasmids was over 10,000,000 clones, exceeding the diversity of the original library by more than an order of magnitude. We then digested the library with NheI and NotI to obtain the N-terminal half of the gene linked with the barcodes. Illumina TruSeq-like adapters were prepared by extension and digestion of two partially complementary oligonucleotides. Libraries with a low complexity reduce the quality of NGS sequencing, thus, we added between one and four random nucleotides after the TruSeq adapters to randomize the beginning of the reads and introduce shifts in the sequence. We ligated the adapters to the libraries by sticky-end ligation.