By Martin Shervington
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RNA cleavages by T1 ribonuclease (T1) are performed in semidenaturing conditions for mapping all G residues. The vertical lines with positions of G residues indicated represent various compositions of T1 fragments that are observed in the presented autoradiograms, derived from precursors with a 5' end heterogeneity (left) and a homogenous sequence (right). F, formamide ladder. 3. Guanosine or adenosine is added to the reaction mixture at the appropriate concentrations to introduce them at the 5' end in a high proportion of transcripts.
Genes Dev. 17, 3011–3016. 36 Schwarz, D. , and Zamore, P. D. (2003) 36. Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199–208. , and Jayasena, S. D. (2003) Functional siRNAs and miRNAs 37. exhibit strand bias. Cell 115, 209–216. , Hamilton, A. , Thomas, C. , Maule, A. , and Baulcombe, D. C. 38. (1999). RNA-DNA interactions and DNA methylation in post-transcriptional gene silencing. Plant Cell 11, 2291–2301. The MicroRNA 17 39 Vaistij, F. , and Baulcombe, D. C. (2002). Spreading of RNA targeting and 39.
50 mg/mL 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt in N,N-dimethyl formamide (Fisher Biotech). 16. ). 17. 5), 100 mM NaCl, and 5 mM MgCl2. 2. 1. Preparing Nuclear Extract 1. 100 15-cm plates of HEK293-derived stable cells expressing FLAG–Drosha. 2. 3. 3.