Blue:RNA-Seq Experiments:11052013

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Purpose[edit]

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


RNAseIII-Fragmented Library Preparation[edit]

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


File:2100 expert Eukaryote Total RNA Pico DE72903057 2013-11-06 12-24-52.pdf

Results:

  • RNaseIII fragmentation is very efficient (fragments centered on about 75-100nt) and doesn't change with increasing MnCl2
  • PAP is not fully complete, and smear is quite high. Seems to decrease in length with increasing MnCl2
  • Substitution of MnCl2 for MgCl2 shows similar smear pattern as unfragmented - indicating MnCl2 works better for RNaseIII

Testing dATP on PAP Activity[edit]

  1. 1mM ATP:0mM dATP (1:0)
  2. 1mM ATP:0.05mM dATP (1:20)
  3. 1mM ATP:0.1mM dATP (1:10)
  4. 1mM ATP:0.2mM dATP (1:5)
  5. 1mM ATP:1mM dATP (1:1)
  6. 0mM ATP:1mM dATP (0:1)


1ul 10uM TSO 1ul 5x ePAP buffer 0.5ul 25mM MnCl2 0.5ul ATP/dATP 1.0 ul 0.4U/ul ePAP 1ul dH2O


File:11052013 gel1.jpg

Results: dATP was able to limit ePAP activity to a small degree when added 1:1 with ATP