Blue:RNA-Seq Experiments:11182013
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Purpose
- Test Affymetrix PAP on TSO poly(A) tailing using similar protocol as 11-13-2013
- Test ability of [dATP] in the RT mix for inhibition of PAP activity
- Test whether RNaseIII retains activity on Poly(A) tailed RNA
RNAseIII-Fragmented Library Preparation
- Conditions
- TSO (no PAP)
- TSO + PAP
- TSO + PAP + 3'dATP
- TSO + PAP + dATP
- TSO + PAP + dNTP (similar conc as RT reaction)
- TSO + RNaseIII + PAP
- TSO + RNaseIII + PAP (no Rnase Inhibitor)
- RNA
Volume 1uM TSO.r04 1ul Total 1ul
- Incubate @ 72C 3 min
- Incubate @ 37C 1 min
Volume 10x RNaseIII buffer or 5x PAP buffer 0.2ul or 0.4ul /- RNase III 0.1ul dH20 0.8ul or 0.6ul Total 2ul
- Incubate @ 37C 5 min
- 3' Tailing
Volume TSO 2ul 5x RT buffer or 5x Affy PAP Buffer 0.5ul or 0.4ul 1mM ATP (or mix) 0.5ul 2U/ul Ambion PAP or 600U/ul Affymetrix PAP 0.2ul 40U/ul Rnase Inhibitor 0.2ul dH2O 0.6ul or 0.7ul /- dNTP mix 0.4ul Total 4ul
- Incubate @ 37C 10 min