Iscript advanced

Tissue panel RNAs were purchased (Clontech, 20 tissue panel II). Three sets of cDNAs were made with a 1:1 mixture of random hexamer and oligo-dT priming with three different RTs: (1) iScript advanced (42 °C, Bio-Rad); (2) Superscript III (55 °C, Invitrogen); and (3) AMV (50 °C, Invitrogen). All RNA samples were spiked with a known amount of MS2 bacteriophage RNA to enable normalization of absolute molecule counts from ddPCR. For tissue panel hTERT and NOVA1 mRNA analysis we used three RTs because we observed differences in detection of hTERT using different RTs so to be able to eliminate spurious measures of low abundance targets we averaged data for all three RTs. All cDNAs were diluted 1:4 before use and stored at −80 °C. For hTERT splicing analyses we used iScript advanced (Bio-Rad) to generate cDNAs, diluted 1:4, and used within 48 h of production in ddPCR measures. Primer sequences for TERT are listed in Supplementary Table 2. Uncropped gel images of RT-PCR gels can be found in Supplementary Figure 7.

cDNA was generated with iScript Advanced (Bio-Rad) or RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific). qPCR reactions using the KAPA-SYBR FAST qPCR kit (Kapa Biosystems) or LightCycler® 480 SYBR Green I Master mix and the primers listed in Supplementary Table 1 were used to measure expression levels on a Bio-Rad CFX96 Touch Real-Time PCR machine or The LightCycler® 480. All primers were tested to ensure the efficiency levels. Expression levels were normalized to those of RP49 and t-tests were used for statistical analysis.

RNA was extracted using RNeasy kit (Qiagen) following the manufacturer’s instructions. cDNA was generated using iScript Advanced (Biorad), and qPCR was set up using PowerUP SYBR green qPCR mastermix (Thermo Fisher) and run on a CFX384 Touch Real-Time PCR detection system (Biorad). Housekeeping genes TBP and/or 18S were used to normalise the data. Primer sequences are listed in Supplementary Table 4.