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
  1. TSO (no PAP)
  2. TSO + PAP
  3. TSO + PAP + 3'dATP
  4. TSO + PAP + dATP
  5. TSO + PAP + dNTP (similar conc as RT reaction)
  6. TSO + RNaseIII + PAP
  7. 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