G 50 microspin columns

MaP reverse transcription was performed as described (20 (link),33 (link)). For both endogenous and plasmid NM_000295, 2 pmol of gene-specific primer (1 μl of 2 μM of primer) was mixed with 1 μg of total RNA for an 8 μl RNA-primer mix (Supplementary Table S6). To RNA-primer mixes, 2 μl of 10 nM dNTPs were added and heated to 68°C for 5 min, and then immediately placed at 4°C for 2 min. To this template solution, 9 μl of freshly-made 2.22× MaP buffer [111 mM Tris (pH 8.0), 167 mM KCl, 22 mM DTT, 6 mM MnCl₂, 2.22 M betaine] was added, and the mixture was incubated at 23°C for 2 min. SuperScript II reverse transcriptase (200 units, Thermo Fisher) was added, and reaction mixtures were incubated at 25°C for 10 min, 42°C for 90 min, 10 × [50°C for 2 min, 42°C for 2 min], and 72°C for 10 min to inactivate enzyme. Reverse transcription reactions were buffer exchanged into TE buffer [10 mM Tris–HCl (pH 8.0), 1 mM EDTA] using G-50 microspin columns (Illustra, GE Healthcare).

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.

Oligonucleotides were purchased from Sigma (Merck, Darmstadt, Germany) (Table S1) and 5′-end labeled by incubation with [³²P]-ATP (Hartmann Analytic, #HP-601-10) (Braunschweig, Germany) and T4 polynucleotide kinase from Thermo Scientific (Paisley Park, UK) for 90 min at 37 °C in 1xforward buffer A from Thermo Scientific (Paisley Park, UK). Reactions were stopped by incubation at 90 °C for 1 min. Free [³²P]-ATP was removed by the use of G50 microspin columns from GE Healthcare (Chicago, IL, USA) according to manufacturer’s protocol. Labeled oligonucleotide was annealed to a partially complementary oligonucleotide in 175 mM KCl and 100 mM EDTA with 5 min incubation at 90 °C, followed by gradual cooling overnight.