Blue:RNA-Seq Experiments:11052013
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Purpose
- Use bioanalyzer to test different fragmentation/poly(A) conditions
RNAseIII-Fragmented Library Preparation
- Conditions
- 5mM MnCl2 (no RNaseIII)
- 5mM MnCl2 (no RNaseIII)/2.5mM MnCl2 (PAP)
- 5mM MnCl2 (RNaseIII)/2.5mM MnCl2 (PAP)
- 5mM MgCl2 (RNaseIII)/2.5mM MgCl2 (PAP)
- 10mM MnCl2 (RNaseIII)/5mM MnCl2 (PAP)
- 10mM MgCl2 (RNaseIII)/5mM MgCl2 (PAP)
- 15mM MnCl2 (RNaseIII)/7.5mM MnCl2 (PAP)
- 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