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Resulting annealed product was end labelled using [γ-32P] ATP as described above.
However, the rNSs removes the α phosphate from the 5' end labelled nucleic acid.
Free nucleotides were removed from the end labelled substrate using Sephadex G25 spun column.
Primers were 5' end labelled with 32PγATP and T4 PNK (NEW England Biolabs).
A Myc-binding E-box containing double-stranded oligonucleotide (supplementary Table S3) was end labelled with 32PdCTP.
U3 snoRNA was detected with 32P 5' end labelled oligonucleotide O202 (5' CCA ACT TGT CAG ACT GCC ATT 3').
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The oligonucleotides were 5'-end labelled with [32P]ATP using T4 polynucleotide kinase (NEB).
20-mer oligonucleotides (see Additional File 3) were 5' P-end labelled with [gammaP]ATP.
The primer was 5′-end labelled with T4 polynucleotide kinase (NEB) and [ γ-P] ATP (5000 Ci mmol−1, Amersham).
To map the cleavage sites in X0 four identical junctions were made, each of which was 5' end-labelled in a different strand.
Alternatively, if cleaving reagents are used, RNAs may be end-labelled and separated by electrophoresis without the reverse transcription step.
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