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As the final method used to validate our predicted IGS exon clusters and assess for differential expression between CX-5461 treated and untreated RNA, we performed a variation of the previous qPCR attempt, but this time accounting for gDNA contamination. To reduce gDNA contamination, we extracted RNA from a smaller input of cells and using the Nucleospin kit. Using less cells is thought to reduce clogging of the column, which reduces the specificity of RNA alone being retained in the column (Liam Williams, University of Auckland, personal communication). The RNA outputs were assessed for gDNA contamination via qPCR with no cDNA synthesis step, using GAPDH primers. Amplification ahead of the water control was observed, suggesting that gDNA was still present in the sample (data not included).

To perform qPCR and assess for IGS exon transcription whilst accounting for gDNA

amplification, we performed qPCR on 500ng cDNA input from CX-5461 treated and untreated samples (in duplicates, cDNA was synthesised from 1 µg of RNA). In parallel to this, we performed qPCR on RNA that wasn’t reverse transcribed (NRT control). The NRT control was made by diluting 1µg of RNA to the cDNA synthesis reaction volume, then using the volume of the diluted RNA proportional to volume of 500ng of cDNA used for qPCR. This is proportional to the amount of residual gDNA in the cDNA samples put into qPCR, and consequently accounts for background gDNA amplification. The primer sets used were Capture-seq exon

Primers 4242DMSO1 4242DMSO2 4242 CX1 4242 CX2 H20

1 3.998392 12.92892 3.335491 4.872675 na 2 6.269595 13.56295 5.2487185 6.659766 na 3 7.0713225 15.57941 6.531418 7.596578 na 4 4.990355 12.09823 4.260963 5.783729 na 5 7.5296105 14.7417 7.3225105 8.027001 33.60457 6 5.7332285 10.30548 5.579123 6.526817 32.31664 7 6.047087 13.31495 5.276643 7.791553 34.42954 8 4.1881435 9.710773 3.834789 5.237281 34.58596 9 4.3941905 12.58312 4.192106 5.588607 32.95678 10 2.440312 8.173154 2.4226655 3.556055 30.68901

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primer sets 12-19, which showed generally better amplified efficiency and produced a single product (by conventional PCR). The target regions of these primer sets can be seen in Figure 20C, as well as in Appendix 1. From the qPCR results and in reference to Figure20 A and B, we saw amplification using the majority of primer sets earlier in cDNA wells than in NRT control (or gDNA background contamination) wells in both CX-5461 treated and DMSO control RNA extractions (figure depicts average of replicates), supporting that there is amplification of cDNA that originates from these predicted exon clusters in the IGS (above background gDNA signal). When assessing all the Ct values (Figure 20D) , for some replicates there are

inconsistencies in Ct values that may reflect pipetting error ( I.e. Set 13 4242 CX-5461 duplicate 2 and Set 18 4242 CX-5461 duplicate 2), that are effecting final normalised results. To address this, samples should be assessed several times with more technical and biological replicates of CX-5461 treated and untreated. Otherwise, most primer sets show consistent patterns of Ct differences between NRT and cDNA samples. Importantly, primer sets with Ct difference consistently higher between cDNA and NRT (primer sets 17, 18, 19 amplifying exon clusters 2, 4, 8) amplifying within predicted exon clusters towards the end of the IGS. We have yet to confirm transcription from exon cluster 5 due to lacking a primer set. In reference to Figure 13, exon clusters 2, 4 and 8 (as well as 5) are in regions with show higher normalised coverage values. We propose that these regions are the most transcriptionally active exon clusters in the IGS. Because different primer sets will have different Ct values, we cannot use this data to confirm which exon clusters are more highly transcribed.

To assess for any potential differential expression of exon clusters between CX-5461 and control DMSO RNA extractions, we briefly compared differences in the average Ct values of the cDNA and gDNA wells between CX-5461 treated and untreated RNA extractions. We class an exon cluster that shows differential expression in the presence or absence of treatment, as a qPCR result that shows limited/no difference between cDNA and gDNA Ct values in one treatment scheme (Ct differences of~0), and an obvious difference in the other (Ct difference of >0). The results can be seen in Figure 20 C. This data does not support that any of the exons show clear differential expression upon CX-5461 treatment. The dramatic difference in set 13 is likely to be a result of pipetting error.

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To briefly assess the extent of the CX-5461 driven inhibition of rRNA synthesis via qPCR, we performed qPCR with primers designed to the 47S pre-rRNA ETS (primer set ETS in Appendix 1). Similar to before, we compared qPCR Ct values of cDNA and underlying gDNA

contamination (by direct RNA input) for 4242 DMSO control and CX-5461 treatment

extractions in duplicates after amplification with the primers, as well as with a water control. The results can be seen in Figure 21 . In terms of the average Ct values, we see cDNA samples amplifying ahead of NRT controls, again suggesting amplification is not solely a reflection of gDNA contamination. Upon normalising to NRT and in reference to Figure 21 B , we see very little difference in Ct values between CX-5461 and control libraries. Consequently, these results suggest that there were limited changes in levels 47S pre-rRNA levels, suggesting inhibition of RNA pol I by CX-5461 was not effective at this time. The small difference of ~0.3 of a cycle is likely to be explained by one of the NRT controls failing for the CX-5461 cycle.

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