Amv rt

All reagents and chemicals unless otherwise noted were obtained from Sigma Aldrich (St. Louis, MO, USA). All oligonucleotides and gBlocks were obtained from Integrated DNA Technologies (IDT, Corralville, IA, USA). The fluorophore-labeled oligonucleotides used for assembling the duplex OSD probe were designed to contain an inverted dT group at their 3’-ends to prevent polymerase-mediated extension (Table 1). oligonucleotides were resuspended at 100 μM concentration in TE (10:0.1, pH 7.5) buffer (10 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, pH 8.0) and stored at -20 °C. The concentrations of the DNA and RNA suspensions were measured by UV spectrophotometry using the NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). All enzymes including Bst 2.0 DNA polymerase and AMV RT were obtained from New England Biolabs (NEB, Ipswich, MA, USA). Human genomic DNA was obtained from Promega (Madison, WI, USA).

1 μg of Turbo DNase-treated RNA was reverse transcribed with the iScript cDNA synthesis kit (BioRad, 1708890), treated with 0.5 μL RNase cocktail (Invitrogen, AM2286), and diluted 1:10. cDNA was quantified using the SensiFAST SYBR No-Rox kit (Bioline, BIO-98005) and 1 μM of each primer (primer sequences provided in Supplementary Table 5). qPCR settings were as follows: 95 °C for 10 min and 40 cycles consisting of 10 s at 95 °C, 20 s at 60 °C, and 20 s at 72 °C, followed by melting curve analysis. TER1 and U6 levels were normalized to act1 mRNA levels and the average wild-type Ct value, and subsequently subject to unpaired two-tailed Student’s t tests and, where applicable, one-way ANOVA followed by a Tukey post hoc test with α set to 0.05 (Supplementary Data 4).
For semi-quantitative RT-PCR, DNase-treated RNA, 10 nmol dNTP mix, and 10 pmol gene-specific reverse primers (Supplementary Table 5) were heated to 65 °C and slow-cooled to 37 °C before reverse transcription with 5 U AMV-RT (NEB, M0277L) at 42 °C for 1 h. cDNA was amplified with Taq polymerase (NEB, MO273L) using standard protocols and the following cycling conditions: 5 min initial denaturation at 94 °C, 22 (TER1) or 17 (U6) cycles of 30 s at 94 °C, 30 s at 57 °C, and 1 min at 72 °C, and a final 10 min extension at 72 °C.

An RNA oligonucleotide (5′-GGCATGTGATTGGTGGGTC) was 5′ labelled with gamma ³²P ATP by T4 polynucleotide kinase (NEB) according to the manufacturer’s instructions. Labelled oligonucleotides were separated from non-incorporated nucleotides via G50 MicroSpin columns (GE Healthcare). One microgramme of total RNA from the indicated gradient fractions was annealed to 0.3 pmol of radiolabelled specific primer in 30 mm Tris-Cl pH 7.5 and 2 mm KCl at 90°C for 2 min and was then cooled down to room temperature for 5 min. Reverse transcription reaction with 0.2 U/μl AMV RT (NEB) was performed at 42°C for 30 min using a dNTP/ddCTP mix at 0.25 mm each (final concentration). Subsequently, RNaseH (Epicentre) was added to the reaction and incubated at 37°C for 10 min to degrade RNA. To 5 μl cDNA, 5 μl RNA loading dye (formamide + bromophenol blue) was added, and RT products were resolved on a 12% denaturing PAGE (7 M urea, 1× TBE, 30 cm). After the run, the gel was fixed and dried on Whatman paper and was exposed to a phosphorimager screen.