Superscript 3 first strand synthesis reagents

RNA was extracted using a RNeasy kit with on column DNase digestion according to the manufacturer's protocol (Qiagen, 74106). 3 μg total RNA (quantified using a NanoDrop spectrophotometer) was used for reverse transcription using SuperScriptIII first-strand synthesis reagents and oligo dT according to the manufacturer's protocol (Invitrogen, 18080051). cDNA was diluted in a final volume of 200 μl nuclease-free water and further diluted one in five prior to amplification, resulting in Ct values in the range of 20–30 cycles. Quantitative PCR was performed using 2× Fast SYBR Green Master mix (Thermo Fisher Scientific, 4385612) in a final volume of 20 μl, using 2 μl cDNA (post one in five dilution). For each biological replicate, individual amplification reactions were performed in technical triplicates, and primers targeting the house-keeping gene Gapdh were included in separate wells for each condition to normalise transcript levels. Primers were designed to span an exon-exon junction using primer-BLAST, and synthesised by Sigma-Aldrich. Sequences can be found in Supplementary Methods Table S5. The StepOne Plus Real Time PCR system (Thermo Fisher Scientific) was used to obtain raw Ct values, and the comparative Ct method 2^–ΔΔCt used to quantify transcript levels.

iSLK BAC16 WT and BAC16 mutant cells were reactivated with doxycycline (1 μg/ml) and RNA was isolated from these cells 0, 24, 48, and 72 h post-induction using RNeasy columns (Qiagen). cDNAs were synthesized using SuperScript III first-strand synthesis reagents (Invitrogen). Gene expression was analyzed by qPCR using specific primers designed by Fakhari and Dittmer^{65 (link)}. KSHV gene expression was normalized to the cellular ACTB signal. RNA harvested from PAN RNA transfected cells included a DNase I treatment step.