Blue:RNA-Seq Experiments:10282013

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Revision as of 18:16, 28 October 2013 by >B1lake (→‎Results of Amplification)
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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 2 min/PAP 5min TSO.r06 T20V.id1-8 N2.id1 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3 2 min/PAP 10min TSO.r06 T20V.id9-16 N2.id2 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3 2 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

  • 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

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

  • Run 5ul on gel:

Final Library Preparation

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