Blue:RNA-Seq Experiments:12032013

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Revision as of 23:56, 11 December 2013 by >B1lake (→‎Final Library Preparation)
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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 1ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP TSO.r06 T20VN_PE_R N2.id81-82 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP TSO.r06 T20VN_PE_R N2.id83-84 totoRNAseq
BL_ UHRR/ERCC 10pg - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP TSO.r06 T20VN_PE_R N2.id385-86 totoRNAseq
BL_ UHRR/ERCC 1ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP TSO.r07 T20VN_PE_R N2.id87-88 totoRNAseq
BL_ UHRR/ERCC 1ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP/One step TSO.r06 T20VN_PE_R N2.id89-90 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP/One step TSO.r06 T20VN_PE_R N2.id91-92 totoRNAseq
BL_ UHRR/ERCC 10pg - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP/One step TSO.r06 T20VN_PE_R N2.id93-94 totoRNAseq
BL_ UHRR/ERCC 1ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP/One step TSO.r07 T20VN_PE_R N2.id95-96 totoRNAseq

Purpose

  • Test efficiency of Affymetrix PAP to generate libraries from decreasing amounts of RNA from 1ng down to 10pg
  • Use ERCCs to determine level of transcript amplification between libraries - ERCC amplification using previous version of the totoRNAseq protocol from 09-27-13 showed progressively lower ERCC quantities in the final libraries from 100pg and 10pg UHRR

RNAseIII-Fragmented Library Preparation

RNA
Volume
UHRR 2ng/ul; 0.2ng/ul; 0.02ng/ul 0.5ul
ERCC 1:10E4; 1:10E5; 1:10E6 0.2ul
0.1uM T20VN 0.1ul
dH2O 0.2ul
Total 1ul


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


Volume
10x RNaseIII buffer 0.2ul
RNase III 0.1ul
dH20 0.7ul
Total 2ul


Incubate @ 37C 5 min


3' Tailing
Volume
fRNA 2ul
5x RT buffer 0.5ul
1mM ATP 0.5ul
600U/ul Affymetrix PAP 0.2ul
40U/ul Rnase Inhibitor 0.2ul
dH2O 0.6ul
Total 4ul


Incubate @ 37C 10 min
Add 1ul of 0.1uM T20VN [for 1-step: 0.1ul 1mM T20VN + 0.9ul 10uM TSO]


Reverse Transcription
Volume
PAP RNA 5ul
5xRT Buffer 1.5ul
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 (2-step only)
  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; vacuspin to 3ul


TSO Reaction (2-step only)
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 (1ng) 12x (100pg) 15x (10pg)
72C, 5min

Results of Amplification

  • Run 5ul on gel:

File:12032013 PCR1 gel1.jpg


Results: Can see that the Affymetrix PAP protocol loses efficiency with decreasing RNA - may not be sensitive enough. R07 on 1ng looks very poor - too inefficient. 1-step does not appear to be an option, r07 in this context does not seem to be affected by PAP, but clearly works poorly for template switching.

Final Library Preparation

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

See 12062013 for gel image of final library (1ng)