Sequagel ureagel system

Turbo DNase-treated total RNAs were incubated with RNase H (New England Biolabs) in the presence or absence of oligo(dT)_12–18. The resulting RNAs were ligated with a 3′ linker, containing a blocked 3′ (ddC) end and an activated adenosine at the 5′ end using truncated T4 RNA ligase 2 (New England Biolabs). RNAs were then purified using a NucleoSpin miRNA kit (Macherey-Nagel) to remove free 3′ linker oligos, followed by reverse transcription using RT-primer that anneals to the 3′ linker sequence. cDNAs were purified with Agencourt AMPure XP beads (Beckman Coulter). The first PCR step was performed with only a gene-specific sense primer for 30 cycles, second PCR amplification was performed with nested sense primer labelled at the 5′ end with [γ-³²P]ATP using T4 polynucleotide kinase (New England Biolabs) and reverse primer annealing to the 3′ linker. Primers are listed in the Supplementary Table 2 (^# indicates primers labelled with ³²P). The resulting PCR products, as well as a 50-bp DNA ladder (Life Technologies) labelled with [γ-³²P]ATP, were loaded on 6% acrylamide gels (SequaGel UreaGel System, National Diagnostics). The gel was then placed on a piece of Whatman filter paper, dried under vacuum and heat (80 °C), exposed and scanned on a Fujifilm Image Reader (Fujifilm FLA 7000).

DNA oligonucleotides were purchased from Integrated DNA Technologies
(IDT) and resuspended in water. Slats of the same type (e.g., x, y,
nuc-x) and of the same length were pooled for combined purification.
Cut slats and nucleic slats of different lengths were individually
purified. Pools were mixed with at least the same volume of 95% formamide,
0.025% (w/v) bromophenol blue, and 5 mM EDTA loading buffer. The SequaGel
UreaGel System (National Diagnostics) was used to prepare 15% denaturing
PAGE gels in empty plastic 1.5 mm minigel cassettes (Invitrogen Novex).
Empty gels were prerun for 1 h at 300 V in 0.5× TBE buffer (45
mM Tris, 45 mM boric acid, 0.78 mM EDTA), and then samples were loaded
and run at 300 V for at least 35 min. Bands of the correct molecular
weight were excised using UV shadowing, crushed with a pestle, and
shaken at 1500 rpm in 500 μL of 1× TE buffer (5 mM Tris
1 mM EDTA) overnight. Extracts were separated by centrifugation in
Freeze N’ Squeeze tubes (Bio-Rad, 732-6166); 2.5–3 volumes
of 100% ice-cold ethanol and 0.1 volume of 3 M sodium acetate were
added to the samples, followed by mixing by inversion and incubation
for 15 min at −80 °C. Samples were precipitated in a refrigerated
centrifuge, washed twice with 70% ice-cold ethanol, dried in air,
and resuspended in water. The final yield was determined by using
a Nanodrop 2000c spectrophotometer (Thermo Scientific).

DNA templates carrying a T7 promoter was PCR-generated from pX330 plasmids using Herculase II Fusion DNA Polymerase (Agilent), ethanol precipitated, and in vitro transcribed with in house made rNTPs, T7 buffer, and T7 polymerase. After 2 h of incubation at 37 °C, 50 U of TurboDNase (Life Technologies) was added and incubated for 30 min at 37 °C. The reactions were stopped with half a volume of formamide loading buffer, and was followed by heat denaturation step for 5 min at 95 °C. Eight percent PAGE-Urea gel was prepared with SequaGel-Urea Gel system (National Diagnostics) and pre-run at 25 W. A total of 400 uL of each samples were loaded, run at 25 W for 1.5 h, visualized with UV lamp set on short wavelength, and gel purified.