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. 5mM MnCl2 (no RNaseIII)
  2. 5mM MnCl2 (no RNaseIII)/2.5mM MnCl2 (PAP)
  3. 5mM MnCl2 (RNaseIII)/2.5mM MnCl2 (PAP)
  4. 5mM MgCl2 (RNaseIII)/2.5mM MgCl2 (PAP)
  5. 10mM MnCl2 (RNaseIII)/5mM MnCl2 (PAP)
  6. 10mM MgCl2 (RNaseIII)/5mM MgCl2 (PAP)
  7. 15mM MnCl2 (RNaseIII)/7.5mM MnCl2 (PAP)
  8. 20mM MnCl2 (RNaseIII)/10mM MnCl2 (PAP)


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
25mM MnCl2 0.08ul
RNase III 0.1ul
dH20 up to 0.83ul
Total 2ul


Incubate @ 37C 5 min
Column Purify


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