Sequencing of octamer library between two SDs

Within a 2 exon mini-gene reporter construct, two splice donor sites of the same intrinsic strength (HBS=17) were inserted at the downstream end of the first exon. To asses the influence of splicing regulatory proteins on splice donor competion, a octamer library (NNNNNNNN) was placed inbetween the two splice sites. The remaining sequence segments, surrounding the octamer library, were selected to be as neutral in their predicted influence on splice site usage as possible, using the HEXplorer tool. The library was sequenced before (plasmid) and after transfecting cells with the reporter and isolating RNA fragments with downstream SD usage by gel-electrophoresis (downstream SD usage). Octamer sequences recruiting SR-proteins would be expected to be enriched in data of downstream_SD_usage, compared to the data of the sequenced plasmid.