A sequence library was designed to include: 63-nucleotide-long wild-type sequence of FAS exon 6, all possible 189 single-nucleotide substitutions, all 2010 possible deletions ranging in length from 1 to 60 nts, all 5208 possible 1-, 2-, and 3-nucleotide-long insertions as well as 400 randomly-selected 4-nucleotide insertions (full library design along with measured PSI values available in Supplementary Table 1). The library was synthesized and purified by Twist Bioscience. Oligo libraries were resuspended in 10 mM Tris buffer, pH 8.0 to a concentration of 20 ng/ul. 20 ng of template ssDNA Twist  library were amplified with Pfx Accuprime polymerase (Thermo scientific, 12344024) in a 50 ul reaction total volume in quadruplicate, for 12 cycles (as recommended by Twist Bioscience for a 100-150 nt oligo pool) using the following flanking intronic primers: FAS_i5_GC_F (5'-tgtccaatgttccaacctacag-3') and FAS_i6_GC_R (5'-ctacttcccaagttatttcaatctg-3'). PCR reactions were combined and cleaned-up with the Quiaquick PCR purification kit, eluted with 50 ul EB buffer and dsDNA measured with a NanoDrop spectrophotometer. The amplified library was recombined with pCMV FAS wt minigene exon 5-6-7 46. We used a vector:insert ratio of 1:8, using 150 ng of vector backbone and 20 ng of dsDNA amplified libraries and incubated at 50ºC for two hours for DNA assembly, using a mix developed at our CRG proteomic facility, which contains a mix of three enzymes an exonuclease, a polymerase and a DNA ligase. After transformation into Stellar competent cells (Clontech, 636766) combining five replicates in order to maximise the number of individual transformants amplified by growing for 18 hours in LB medium containing ampicillin. We obtained approximately 4.29 million clones. After bacterial transformation, the final plasmid library was purified using the Quiagen plasmid maxi kit (50912163, Quiagen) and quantified with a NanoDrop spectrophotometer. 750,000 Hek293 cells were plated on 100x20 mm petri dishes and transfected with 80 ng cloned libraries in 8 ml OPTIMEM Reduced Serum Medium with no phenol Red (Life technologies, 11058021) using Lipofectamine 2000 (Life technologies, 11668019) in nine biological replicates. 48h post transfection, cells were collected and RNA was prepared using Maxwell simplyRNA Tissue Kit (Promega, AS1280). cDNA was prepared with 400 ng total RNA using specific vector backbone PT2 primer (5'- AAGCTTGCATCGAATCAGTAG -3') and Superscript III reverse transcriptase (Thermo Fisher, 18080085). PCR amplification of cDNA samples was performed with GoTaq flexi (PROMEGA, M7806) using a distinct 8-mer barcoded oligos to distinguish the nine experimental replicates. PCR products were run on a 2% agarose gel and the smear corresponding in sizes to the amplification product of exon inclusion was excised, purified using the Quiaquick Gel extraction kit (Quiagen, 50928704) and quantified with a NanoDrop spectrophotometer. OTHER: DNA seq of PCR amplicon