Title-NOVA1 directs PTBP1 to hTERT pre-mRNA and promotes telomerase activity in cancer cells.

We used long read sequencing in this study to determine the splice isoform diversity of the TERT (telomerase reverse transcriptase) gene from two species (humans and mouse). The mouse cDNA libraries were generated using a mouse Tert specific primer in the final exon the gene. cDNA libraries were generated with three different reverse transcriptases (Bio-Rad iscript advanced, superscript 3 and AMV). Then barcoded primers were used to PCR amplify the mTert specific cDNAs. Amplicons were gel purified and pooled at equal concentrations and sent to PacBio for SMRT library prep and sequencing on the Pacific Biosciences real-time sequencer using C2 sequencing reagents with 90-min movies. For the human samples, we generated two different preparation schemes. In one scheme we, we generated gene specific cDNAs as above and then pulled down TERT from exon 1 and performed PCR (with barcoded primers). Following PCR the amplicons were gel purified according to size. Either above 2.5 kb or below 2.5 kb. The longer fragments were pooled and sequenced in 3 lanes of a SMRT cell and the lower bands were sequenced on 1 lane of SMRT cell. In the second scheme we used the same cDNAs and generated PCR amplicons using primers in exon2 and exon 16 of hTERT. Amplicons were barcoded, size selected, and sequenced as above.