Blue:RNA-Seq Experiments:11132013

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Revision as of 21:41, 14 November 2013 by >B1lake (→‎RNA-Seq Library Information)
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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 5ng - RNase3 (RIII buffer)/Ambion PAP 10min ATP TSO.r06 T20VN_PE_R N2.id90 totoRNAseq
BL_ UHRR/ERCC 5ng - RNase3 (RIII buffer)/Ambion PAP 10min ATP/3'dATP (1:0.1) TSO.r06 T20VN_PE_R N2.id91 totoRNAseq
BL_ UHRR/ERCC 5ng - RNase3 (RIII buffer)/Ambion PAP 10min ATP/dATP (1:1) TSO.r06 T20VN_PE_R N2.id92 totoRNAseq
BL_ UHRR/ERCC 5ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP TSO.r06 T20VN_PE_R N2.id93 totoRNAseq
BL_ UHRR/ERCC 5ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP/3'dATP (1:0.1) TSO.r06 T20VN_PE_R N2.id94 totoRNAseq
BL_ UHRR/ERCC 5ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP/dATP (1:1) TSO.r06 T20VN_PE_R N2.id95 totoRNAseq
BL_ UHRR/ERCC 5ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP then add 3'dATP TSO.r06 T20VN_PE_R N2.id96 totoRNAseq
BL_ UHRR/ERCC 5ng - RNase3 (Affy buffer)/Affymetrix PAP 10min ATP then add 3'dATP TSO.r06 T20VN_PE_R N2.id97 totoRNAseq

Purpose

  • Test original totoRNAseq protocol (using RNaseIII buffer and 5xRT for RnaseIII/PAP treatment) without heating to 65C to inactivate enzymes. RNaseIII inactivation by RNase inhibitor addition and PAP limitation by 3'dATP or dATP addition
  • Testing difference of Ambion PAP and Affymetrix PAP (note that Affymetrix PAP showed significant A tailing of TSO and was completely inhibited by 3'dATP)

RNAseIII-Fragmented Library Preparation

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
10x RNaseIII buffer or 5x PAP buffer 0.2ul or 0.4ul
RNase III 0.1ul
dH20 0.7ul or 0.5ul
Total 2ul


Incubate @ 37C 5 min


3' Tailing
Volume
fRNA 2ul
5x RT buffer or 5x Affy PAP Buffer 0.5ul or 0.4ul
1mM ATP (or mix) 0.5ul
2U/ul PAP 0.2ul
40U/ul Rnase Inhibitor 0.2ul
dH2O 0.6ul or 0.7ul
Total 4ul


Incubate @ 37C 10 min
Add 1ul of 1uM T20VN or 0.5ul 2uM T20VN/0.05uM 3'dATP


Reverse Transcription
Volume
PAP RNA 5ul (or 4.5ul)
5xRT Buffer 1.5ul (or 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 6ul 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 9x
72C, 5min

Results of Amplification

  • Run 5ul on gel:

Final Library Preparation

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