Blue:RNA-Seq Experiments:11052013

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Purpose

  • Use bioanalyzer to test different fragmentation/poly(A) conditions


RNAseIII-Fragmented Library Preparation

Conditions
  1. No RNase III (5mM MnCl2)/no PAP treatment
  2. RNaseIII (5mM MnCl2)/no PAP treatment
  3. RNaseIII (5mM MgCl2)/no PAP treatment
  4. RNaseIII (10mM MnCl2)/no PAP treatment
  5. RNaseIII (15mM MnCl2)/no PAP treatment
  6. RNaseIII (20mM MnCl2)/no PAP treatment
  7. No RNase III (5mM MnCl2)/PAP (2.5mM MnCl2)
  8. RNaseIII (5mM MnCl2)/PAP (2.5mM MnCl2)
  9. RNaseIII (5mM MgCl2)/PAP (2.5mM MgCl2)
  10. RNaseIII (10mM MnCl2)/PAP (5mM MnCl2)
  11. RNaseIII (15mM MnCl2)/PAP (7.5mM MnCl2)
  12. RNaseIII (20mM MnCl2)/PAP (10mM MnCl2)


RNA
Volume
UHRR 10ng/ul 1ul
5x Ambion PAP buffer 0.08ul
dH20 0.09ul
Total 1.17ul


Incubate @ 72C 3 min
Incubate @ 37C 1min


RNA FRAGMENTATION
Volume
RNA 1.17ul
MnCl2/MgCl2 0.4ul
RNase III 0.1ul
dH20 0.33ul
Total 2ul


Incubate @ 37C 5 min
Column Purify Samples 1-6


3' Tailing
Volume
fRNA 2ul
5x Ambion PAP Buffer 0.72ul
ATP (or mix) 0.25ul
2U/ul PAP 0.2ul
40U/ul Rnase Inhibitor 0.2ul
dH2O 0.63ul


Incubate @ 37C 10 min
Column Purify Samples 7-12