Blue:RNA-Seq Experiments:11012013

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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(.1U) 1mM MnCl2 inactivated with Rnase Inhib/PAP 10min ATP TSO.r06 T20V.id2 N2.id81 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3(.01U) 1mM MnCl2 inactivated with Rnase Inhib/PAP 10min ATP TSO.r06 T20V.id2 N2.id82 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3(.001U) 1mM MnCl2 inactivated with Rnase Inhib/PAP 10min ATP TSO.r06 T20V.id2 N2.id83 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3(0U) 1mM MnCl2 inactivated with Rnase Inhib/PAP 10min ATP TSO.r06 T20V.id2 N2.id84 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3(.1U) 1mM MnCl2 inactivated with Rnase Inhib/PAP 10min 3'dATP TSO.r06 T20V.id2 N2.id85 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3(.01U) 1mM MnCl2 inactivated with Rnase Inhib/PAP 10min 3'dATP TSO.r06 T20V.id2 N2.id86 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3(.001U) 1mM MnCl2 inactivated with Rnase Inhib/PAP 10min 3'dATP TSO.r06 T20V.id2 N2.id87 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3(0U) 1mM MnCl2 inactivated with Rnase Inhib/PAP 10min 3'dATP TSO.r06 T20V.id2 N2.id88 totoRNAseq


Note: ATP Condtions:

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

Note: All conditions repeated for 2mM MnCl2 (N2.id89-96)

Purpose[edit]

  • Continuation of tests from 10-30-2013
  • To try and reduce the number of PolyA reads in the totoRNAseq library that might arise from overfragmentation or overactive PAP
    • Reduce chemically induced fragmentation by replacing 65C RnaseIII inactivation with RNase Inhibitor
    • Eliminate all RNA heating steps involving buffer
    • 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?

RNAseIII-Fragmented Library Preparation[edit]

RNA
Volume
UHRR 200pg/ul 0.5ul
ERCC 1:10E5 0.2ul
0.1uM T20V.id2 0.1ul
5x Ambion PAP buffer 0.08ul
dH20 0.29ul
Total 1.17ul
Incubate @ 72C 3 min
Incubate @ 37C 1min


RNA FRAGMENTATION
Volume
RNA 1.17ul
25mM MnCl2 0.08ul
RNase III 0.1ul or 0.01ul or 0.001ul or 0ul
dH20 up to 0.83ul
Total 2ul


Incubate @ 37C 5 min


3' Tailing
Volume
fRNA 2ul
5x Ambion PAP Buffer 0.72ul
25mM MnCl2 0.32ul
ATP (or mix) 0.25ul
2U/ul PAP 0.2ul
40U/ul Rnase Inhibitor 0.2ul
dH2O 0.31ul


Incubate @ 37C 10 min
Transfer to Ice


Reverse Transcription
Volume
PAP RNA 4ul
10mM dNTP Mix 1ul
0.2uM T20V.ID 0.5ul
5xRT Buffer 2ul
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 - 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[edit]

  • Run 5ul on gel:

File:11012013 PCR1 gel1.jpg


Results:

  1. RNaseIII does not seem to be active in the PAP buffer conditions - neither 1mM nor 2mM MnCl2
  2. Elimination of heat inactivation steps seems to prevent over-fragmentation

Final Library Preparation[edit]

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