5′ RACE and 3′ RACE indicate antisense lncRNAs in the integration site in the C5 clone. (A) 5′ RACE PCR to detect the transcription of antisense lncRNA C5-AS1 into the lentiviral integration site of clone C5. C5-AS1 originates from the adjacent genomic region 0.7 kb downstream from the 3′ terminus of the integrated cassette. CIP/TAP/Ligation (left lane) total RNAs were treated by calf-intestinal alkaline phosphatase (CIP), tobacco acid pyrophosphatase (TAP), and ligated to RNA adaptors before analysis by nested PCR. Only lncRNAs with an intact 5′ cap structure can be detected by this method. Ligation (middle lane) total RNAs were treated by CIP and TAP sequentially before they were ligated to RNA adaptor and subjected to nested PCR analysis. The lncRNAs with monophosphate can be detected by this method. CIP/Ligation (right lane) total RNAs were treated by CIP, ligated to RNA adaptors, and analyzed by nested PCR. This lane shows the general background of 5′ RACE. The gel bands indicate lengths of nested PCR products, not the absolute lengths of antisense RNA. (B) 3′ RACE PCR to detect the antisense lncRNAs that are transcribed into the reporter gene cassettes. The total RNAs were reverse transcribed by the adaptor-oligo(dT) primer (left lane). The total RNAs were first tailed with poly(A) sequence before reverse transcription (right lane). The RT products were analyzed by nested PCR and sequencing. Multiple termini were detected, as indicated by the colored arrows. The bands on the gel indicate lengths of nested PCR products, not the absolute lengths of antisense RNA. (C) Schematic diagram shows that a single 5′ terminus (5′ RACE, bottom) and multiple 3′ termini (3′ RACE, upper) and were detected for the antisense lncRNAs that are transcribed into the integration site in C5 clone. In the upper section, the colored arrows represent the 3′ termini of the antisense lncRNAs that were mapped within the reporter cassette. For each of the five lncRNA 3′ termini detected in B, the distance from the 3′ termini of the lncRNA to the 3′ termini of the reporter cassette are indicated. In the lower section, the 5′ terminus of C5-AS1 was mapped within the genomic region downstream from the reporter gene cassette. The distance from the antisense lncRNA starting site to the 3′ terminus of the reporter cassette is indicated.
