Blue:RNA-Seq Experiments:10302013: Difference between revisions

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Result:  RNaseIII concentration does not seem to matter... fragmentation is occurring anyway, indicating 65C enzyme inactivation steps are degrading the RNA.  Need to re-optimize fragmentation step.  Like there are two issues:
Result:  RNaseIII concentration does not seem to matter... fragmentation is occurring anyway, indicating 65C enzyme inactivation steps are degrading the RNA.  Need to re-optimize fragmentation step.  Likely there are two issues:
#RNase III is still not working optimally and the majority of the fragmentation is occurring from Mn-dependent fragmentation
#RNase III is still not working optimally and the majority of the fragmentation is occurring from Mn-dependent fragmentation
#RNase III is fine, but already fragmented RNA is becoming further fragmented (prior to or after PAP - either way reducing poly(A)-tailed fragments for RT)
#RNase III is fine, but already fragmented RNA is becoming further fragmented (prior to or after PAP - either way reducing poly(A)-tailed fragments for RT)

Revision as of 21:33, 31 October 2013

RNA-Seq Library Information

Library ID Samples Input RNA TSO Primer Bar Code Primers Bar Code Primer 2 Type of Seq Reads Library Conc
BL_ UHRR/ERCC 100pg - RNase3(.08U) 5 min/PAP 10min TSO.r06 T20V.id2 N2.id81 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3(.008U) 5 min/PAP 10min TSO.r06 T20V.id2 N2.id82 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3(.001U) 5 min/PAP 10min TSO.r06 T20V.id2 N2.id83 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3(0U) 5 min/PAP 10min TSO.r06 T20V.id2 N2.id84 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3(.08U) 5 min/PAP 30min TSO.r06 T20V.id2 N2.id85 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3(.008U) 5 min/PAP 30min TSO.r06 T20V.id2 N2.id86 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3(.001U) 5 min/PAP 30min TSO.r06 T20V.id2 N2.id97 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3(0U) 5 min/PAP 30min TSO.r06 T20V.id2 N2.id88 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3(.08U) 5 min/PAP 10min/1:0.05 ATP TSO.r06 T20V.id2 N2.id89 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3(.008U) 5 min/PAP 10min/1:0.05 ATP TSO.r06 T20V.id2 N2.id90 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3(.001U) 5 min/PAP 10min/1:0.05 ATP TSO.r06 T20V.id2 N2.id91 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3(0U) 5 min/PAP 10min/1:0.05 ATP TSO.r06 T20V.id2 N2.id92 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3(.08U) 5 min/PAP 30min/1:0.05 ATP TSO.r06 T20V.id2 N2.id93 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3(.008U) 5 min/PAP 30min/1:0.05 ATP TSO.r06 T20V.id2 N2.id94 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3(.001U) 5 min/PAP 30min/1:0.05 ATP TSO.r06 T20V.id2 N2.id95 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3(0U) 5 min/PAP 30min/1:0.05 ATP TSO.r06 T20V.id2 N2.id96 totoRNAseq


Note: ATP Condtions:

  1. 1mM ATP/0mM 3'dATP
  2. 1mM ATP/0.05mM 3'dATP

Purpose

  • Continuation of tests from 10-23-2013 and 10-29-2013
  • To try and reduce the number of PolyA reads in the totoRNAseq library that might arise from overfragmentation or overactive PAP
    • Reduce [RNaseIII] to get less or no fragmentation and and see how this affects the resultant post-PCR amplification
    • Test termination of PAP activity to limit poly-A tail length by adding Cordycepin-ATP or 3'-ATP - can the ~200bp band get reduced by decreasing amount of fragmentation?
    • Include PAP inactivation - heating


RNAseIII-Fragmented Library Preparation

RNA
Volume
UHRR 200pg/ul 0.5ul
ERCC 1:10E5 0.2ul
5x Ambion PAP buffer 0.08ul
0.1uM T20V.id2 0.1ul
25mM MnCl2 0.08ul
dH20 0.29ul
Total 1.25ul
Incubate @ 72C 3 min
Incubate @ 37C 1min


RNA FRAGMENTATION
Volume
RNA 1.25ul
RNase III 0.08ul or 0.008ul or 0.001ul or 0ul
dH20 up to 0.75ul
Total 2ul


Incubate @ 37C 5 min
Incubate @ 65C 10 min



3' Tailing
Volume
fRNA 2ul
5x Ambion PAP Buffer 0.72ul
25mM MnCl2 0.32ul
ATP (or mix) 0.25ul
0.4U/ul PAP 0.2ul
dH2O 0.51ul


Incubate @ 37C 10 min or 30 min
Transfer to Ice


Reverse Transcription
Volume
PAP RNA 4ul
10mM dNTP Mix 1ul
0.2uM T20V.ID 0.5ul


Incubate @ 65C 10 min
Transfer to Ice


Volume
5xRT Buffer 2ul
100mM DTT 0.17ul
Betaine (5M) 2ul
RNase Inhibitor (40U/ul) 0.1ul
Smarter MMLV (200U/ul) 0.23ul
Total 10ul


Incubate @ 42C 30 min


Beads Purification
  1. Add 15ul beads per well
  2. Allow to bind 15 min - combine sets
  3. Remove supernatant and wash 2x with 1ml 80% EtOH
  4. Air Dry 10min or until cracking first occurs
  5. Resuspend beads in 3ul dH2O, incubate 2min, transfer to new tube


TSO
Volume
Purified cDNA 3ul
5x RT buffer [MMLV] 2ul
10mM dNTP mix 1ul
100mM DTT 0.25ul
10uM TSO 1ul
Betaine (5M) 2ul
RNase Inhibitor 0.25ul
Smarter MMLV 0.5ul
Total 10ul


Incubate @42C 10min
Incubate @70C 10min


PCR (PhusionHF)
Volume
TSO Reaction 10ul
2x Buffer 25ul
10uM P1-STRT 1ul
10uM PCR_R_N2_id 1ul
H2O 13ul
Total 50ul


98C 30 sec
98C 15s -> 58C 20s -> 72C 30s 6x
98C 10s -> 72C 20s 12x
72C, 5min

Results of Amplification

  • Run 5ul on gel:

File:10302013 PCR1 gel1.jpg


Result: RNaseIII concentration does not seem to matter... fragmentation is occurring anyway, indicating 65C enzyme inactivation steps are degrading the RNA. Need to re-optimize fragmentation step. Likely there are two issues:

  1. RNase III is still not working optimally and the majority of the fragmentation is occurring from Mn-dependent fragmentation
  2. RNase III is fine, but already fragmented RNA is becoming further fragmented (prior to or after PAP - either way reducing poly(A)-tailed fragments for RT)

Need to ensure RNase III is working and reduce opportunities for chemically induced fragmentation!

Final Library Preparation

  • Beads purified twice at (0.8x), re-suspend in 10ul