Poly a polymerase

Reverse transcription from a linker molecule (which we achieve using polyA polymerase to attach a polyA tail) is necessary for efficient reverse transcription of short, prematurely-terminated HIV transcripts limited to the TAR loop [31 (link)]. Therefore, a polyadenylation step was employed prior to reverse transcription and ddPCR for the TAR region [27 (link), 31 (link)]. Each polyadenylation reaction comprised cellular RNA with 3μL of 10x Superscript III buffer (Invitrogen), 3μL of 50mM MgCl₂, 1μL of 10mM ATP (Epicentre), 2μL of 4U/μL poly-A polymerase (Epicentre), and 1μL of 40U/μL RNaseOUT (Invitrogen) in a 20μL reaction. The reaction was incubated at 37μC for 45min prior to addition of RT reaction components, including 1.5μL of 10mM dNTPs (Invitrogen), 1.5μL of 50ng/μL random hexamers (Invitrogen), 1.5μL of 50μM oligo dT15, and 1μL of 200U/μL Superscript III reverse transcriptase (Invitrogen). Reverse transcription was performed on the final 30μL reaction at 25.0°C for 10 min, 50.0°C for 50 min, followed by an inactivation step at 85.0°C for 5 min.

To generate the T7-VEE-mCherry construct, T7-VEE-OKS-iG (#58974; Addgene, Cambridge, MA, USA) was linearized by NdeI/NotI digestion and modified by removing the OKS-iG sequence [42 ]. This vector was renamed as T7-VEE. pSicoR-Ef1a-mCh-Puro (#31845; Addgene) was digested with the same restriction enzymes, and then the mCherry-Puro sequence was introduced to the T7-VEE vector. The pTNT-B18R vector (#58978; Addgene) was amplified by PCR, and the amplicon was purified by gel extraction. These linearized DNA templates were used for in vitro transcription. The synthesis of T7-VEE-mCherry and pTNT-B18R RNA was performed with the RiboMAX Large Scale RNA Production System-T7 Kit (Promega, Madison, WI, USA). The ScriptCap m7G Capping System (Epicentre, Madison, WI, USA) was used for 5′ capping, which confers mRNA stability and efficient translation. After the 5′ mRNA- capping reaction, a poly (A) tail was added using poly (A) polymerase (Epicentre). These individual RNAs were purified by ammonium acetate and isopropanol precipitation, resuspended in the RNase-free water, and stored at −80 °C. A PCR was performed to confirm that both mRNAs were properly synthesized.