Blue:RNA-Seq Experiments:11122013

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RNA-Seq Library Information[edit]

Library ID Samples Input RNA TSO Primer Bar Code Primers Bar Code Primer 2 Type of Seq Reads Library Conc
BL_ UHRR/ERCC 10ng - RNase3 (5mM MnCl2)/PAP (2.5mM MnCl2) 10min ATP TSO.r06 T20VN_PE_R N2.id81 totoRNAseq
BL_ UHRR/ERCC 10ng - RNase3 (5mM MnCl2)/PAP (2.5mM MnCl2) 10min ATP/dATP TSO.r06 T20VN_PE_R N2.id82 totoRNAseq
BL_ UHRR/ERCC 10ng - RNase3 (5mM MnCl2)/PAP (2.5mM MnCl2) 30min ATP/dATP TSO.r06 T20VN_PE_R N2.id83 totoRNAseq
BL_ UHRR/ERCC 10ng - RNase3 (10mM MnCl2)/PAP (5mM MnCl2) 10min ATP TSO.r06 T20VN_PE_R N2.id84 totoRNAseq
BL_ UHRR/ERCC 10ng - RNase3 (10mM MnCl2)/PAP (5mM MnCl2) 10min ATP/dATP TSO.r06 T20VN_PE_R N2.id85 totoRNAseq
BL_ UHRR/ERCC 10ng - RNase3 (10mM MnCl2)/PAP (5mM MnCl2) 30min ATP/dATP TSO.r06 T20VN_PE_R N2.id86 totoRNAseq
BL_ UHRR/ERCC 10ng - RNase3 (20mM MnCl2)/PAP (10mM MnCl2) 10min ATP TSO.r06 T20VN_PE_R N2.id87 totoRNAseq
BL_ UHRR/ERCC 10ng - RNase3 (20mM MnCl2)/PAP (10mM MnCl2) 10min ATP/dATP TSO.r06 T20VN_PE_R N2.id88 totoRNAseq
BL_ UHRR/ERCC 10ng - RNase3 (20mM MnCl2)/PAP (10mM MnCl2) 30min ATP/dATP TSO.r06 T20VN_PE_R N2.id89 totoRNAseq

Purpose[edit]

  • Full totoRNAseq protocol on samples prepared similarly to 11-06-2013
  • Determine optimal PAP conditions for library production
  • Use T20VN to limit the primer annealing to the ends flanking RNA fragments rather than randomly along extensive poly(A) tails

RNAseIII-Fragmented Library Preparation[edit]

RNA
Volume
UHRR 20ng/ul 0.5ul
ERCC 1:10E3 0.2ul
1uM T20VN 0.1ul
dH2O 0.2ul
Total 1ul


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


Volume
5x Ambion PAP buffer 0.08ul
MnCl2 0.4ul
RNase III 0.1ul
dH20 0.42ul
Total 2ul


Incubate @ 37C 5 min


3' Tailing
Volume
fRNA 2ul
5x Ambion PAP Buffer 0.72ul
1mM ATP (or mix) 0.25ul
2U/ul PAP 0.2ul
40U/ul Rnase Inhibitor 0.2ul
dH2O 0.63ul
Total 4ul


Incubate @ 37C 10 min - 30 min
Add 0.5ul of 2uM T20VN


Reverse Transcription
Volume
PAP RNA 4.5ul
5xRT Buffer 2ul
10mM dNTP Mix 1ul
100mM DTT 0.25ul
Betaine (5M) 2ul
Smarter MMLV (200U/ul) 0.25ul
Total 10ul


Incubate @ 42C 30 min


Beads Purification
  1. Add 15ul beads per well
  2. Allow to bind 15 min
  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 8x
72C, 5min

Results of Amplification[edit]

  • Run 5ul on gel:

File:11122013 PCR1 gel1.jpg


  • Re-Amplified 1ul using ILMN_PCR_F/R and SYBR
  • Ran 5ul on gel:

File:11122013 PCR2 gel1.jpg


Results: Looks like there is ultimately a problem with the altered protocol. It's possible that while MnCl2 allows for more PAP control, it inhibits subsequent RT reactions. The original PCR smear appears too high and upon re-amplification it drops quite low... This similarly high smear was also observed on 11012013 - at that time I miss-interpreted the results as indicating a lack of fragmentation, but instead I now think that the MnCl2 might be blocking subsequent steps. Its possible that rather than focusing on controlling PAP, we should instead focus on preventing accidental fragmentation of poly(A) tailed RNA (i.e. no heating prior to RT reaction).

Final Library Preparation[edit]

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