Blue:RNA-Seq Experiments:10282013

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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 100pg - RNase3 2.5 min/PAP 5min TSO.r06 T20V.id1-8 N2.id1 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3 2.5 min/PAP 10min TSO.r06 T20V.id9-16 N2.id2 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3 2.5 min/PAP 30min TSO.r06 T20V.id17-24 N2.id3 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3 5 min/PAP 5min TSO.r06 T20V.id25-32 N2.id4 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3 5 min/PAP 10min TSO.r06 T20V.id33-40 N2.id5 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3 5 min/PAP 30min TSO.r06 T20V.id41-48 N2.id6 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3 10 min/PAP 5min TSO.r06 T20V.id49-56 N2.id7 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3 10 min/PAP 10min TSO.r06 T20V.id57-64 N2.id8 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3 10 min/PAP 30min TSO.r06 T20V.id64-72 N2.id9 totoRNAseq


Note: Each condition includes 8 ATP variables:

  1. 1mM ATP/0mM 3'dATP
  2. 1mM ATP/0.01mM 3'dATP
  3. 1mM ATP/0.05mM 3'dATP
  4. 1mM ATP/0.1mM 3'dATP
  5. 0.1mM ATP/0mM 3'dATP
  6. 0.1mM ATP/0.001mM 3'dATP
  7. 0.1mM ATP/0.005mM 3'dATP
  8. 0.1mM ATP/0.01mM 3'dATP

Purpose[edit]

  • To try and reduce the number of PolyA reads in the totoRNAseq library that might arise from overfragmentation or overactive PAP
    • Test RNaseIII fragmentation times - want time that doesn't overfragment but gives sharper distribution in sizes around 100-200 bases
    • Test reducing ATP to more limiting 0.1mM
    • Test termination of PAP activity to limit poly-A tail length by adding Cordycepin-ATP or 3'-ATP at different ratios to regular ATP
    • Test PAP incubation times - does longer PAP treatment lead to an increase in lower copy transcripts?
    • Include PAP inactivation - heating


RNAseIII-Fragmented Library Preparation[edit]

RNA
Volume
UHRR 200pg/ul 0.5ul
ERCC 1:10E5 0.2ul
5x Ambion PAP buffer 0.08ul
0.1uM T20V.id 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
dH20 0.67ul
Total 2ul


Incubate @ 37C 2-10 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 5-30 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 10ul dH2O, incubate 2min, transfer to new tube - vacuspin 8 min to bring to 3ul


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 9x
72C, 5min

Results of Amplification[edit]

  • Run 5ul on gel:

File:10282013 PCR1 gel1.jpg


Note: Looks like the RNaseIII might be over fragmenting to generate pooling of small fragments around 200bp. Increasing PAP time might be causing the shift of these up to HMW smear. This would indicate that over fragmentation followed by over polyA tailing would generate the majority of the smear. Need to sequence to know for sure. Need to repeat and separate out the ATP conditions so that each can be observed individually...

Final Library Preparation[edit]

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