Fig 3. Selection of PAN RNA sequences for use in CHART.

(A) Schematic of KSHV PAN RNA indicating the location of known RNA elements MRE (Mta-response element) [57] and ENE (element for nuclear expression) [16], as well as 12 complementary oligonucleotides (A to L) tested for their RNaseH-targeting activity. The overlapping open reading frame K7 is indicated. (B) BCBL-1 cells were induced with 600 μM valproic acid for the indicated time (latent is 0 h); then RNA was harvested. RNA levels of KSHV PAN RNA, KSHV ORF71-72 latent transcript and RNaseP RNA were analyzed by Northern blot. Quantification of three biological replicates normalized to RNase P RNA is shown. (C) Representative RNaseH assays of PAN RNA in BCBL-1 nuclear lysates assessed by Northern blot. No difference in cleavage pattern was observed for the three time points tested. NS: non-specific oligonucleotide complementary to GFP mRNA. (D) Representative enrichment of PAN RNA following CHART RNA isolation with two distinct oligonucleotide sets (blue: oligonucleotides E and K, orange: oligonucleotides F and L). PAN RNA is observed as two bands on a Northern blot following the sonication step of the CHART procedure. Relative to input, approximately 20% of PAN RNA was isolated at each time point. The small fraction of PAN RNA isolated from the latent sample was generated by the 1–3% of cells undergoing spontaneous lytic reactivation. (E) Schematic of RRV PAN RNA indicating the location of the ENE, as well as 3 complementary oligonucleotides tested for their RNaseH-targeting activity. (F) BJAB RRV cells were induced with 500 nM TSA for the indicated time (latent is 0 h); then RNA was harvested. RNA levels of RRV PAN RNA and RNaseP RNA were analyzed by Northern blot. Quantification of three replicates is shown. (G) Representative RNaseH assays of PAN RNA in BCBL-1 nuclear lysates assessed by Northern blot. Cleavage products are indicated by ●. (H) Representative enrichment of RRV PAN RNA following CHART RNA isolation with two distinct oligonucleotide sets (blue: oligonucleotides TKV440 and TKV539, orange: oligonucleotide KT493). PAN RNA is observed as two bands on a Northern blot following the sonication step of the CHART procedure. Quantification of PAN RNA isolated during the CHART procedure is indicated below the Northern blot. S: supernatant P: pellet.