Sequencing of octamer library downstream of an SA

For the generation of the octamer library, a PCR fragment was generated with a primer containing a random 8-mer. PCR fragments were inserted into the respective backbone resulting in a three-exon mini-gene reporter with an octamer library downstream of the splice acceptor of the middle exon. The plasmid library was amplified after transformation of E. coli. The library containing plasmids were then used for transfection, followed by RNA isolation and analysis via RT-PCR using primers and subsequent PAA-gel analysis. For amplicon sequencing, the desired band was excised and purified via the QIAquick Gel Extraction Kit and re-amplified using the same primers. NGS amplicon sequencing was carried out by Eurofins Genomics, Konstanz, Germany using Illumina NovaSeq 6000 PE150 leading to 14,379,943 reads for the plasmid and 8,633,229 reads for the band sample.