Blue:RNA-Seq Experiments:12062013

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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_totoRNAseq_1ng_100pgUHRR_Dec06 UHRR/ERCC 1ng - RNase3 1min (RIII buffer)/Affymetrix PAP 10min ATP TSO.r06 T20VN_PE_R N2.id9-10 totoRNAseq
UHRR/ERCC 100pg - RNase3 1min (RIII buffer)/Affymetrix PAP 10min ATP TSO.r06 T20VN_PE_R N2.id11-12 totoRNAseq
UHRR/ERCC 10pg - RNase3 1min (RIII buffer)/Affymetrix PAP 10min ATP TSO.r06 T20VN_PE_R N2.id13-14 totoRNAseq
UHRR/ERCC 1ng - RNase3 1min (RIII buffer)/Affymetrix PAP 30min ATP TSO.r06 T20VN_PE_R N2.id17-18 totoRNAseq
UHRR/ERCC 100pg - RNase3 1min (RIII buffer)/Affymetrix PAP 30min ATP TSO.r06 T20VN_PE_R N2.id19-20 totoRNAseq
UHRR/ERCC 10pg - RNase3 1min (RIII buffer)/Affymetrix PAP 30min ATP TSO.r06 T20VN_PE_R N2.id21-22 totoRNAseq

Primer sequences [1]

Purpose[edit]

  • Test efficiency of Affymetrix PAP to generate libraries from decreasing amounts of RNA from 1ng down to 10pg as done in 12032013, but with less fragmentation by RNaseIII (1 min vs. 5 min) and longer PAP treatment
  • 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[edit]

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 1 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 10-30 min
Add 1ul of 0.1uM T20VN


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
  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
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[edit]

  • Run 5ul on gel:

File:12062013 PCR1 gel1.jpg


Results: Can see better amplification of 100pg and 10pg after shortening the RNAse III digestion time (compared to 12032013). Increasing PAP time may not be necessary.

Final Library Preparation[edit]

  • Beads purified twice at (0.8x), re-suspended in 10ul
  • Test ERCC amplification:
    • Diluted PCR product 1:5 and used 1ul:
      • PCR Product: 1ul
      • 2x Sybr: 12.5ul
      • ILMN_PCR_F: 0.5ul
      • ERCC130R227 or R964: 0.5ul
      • dH2O: 10.5ul
    • PCR Results:

File:12062013 PCR2 ERCC-3.jpg File:12062013 PCR2 ERCC-5.jpg

Results: Can see that the reactions for 1ng and 100pg show similar efficiency for amplifying the 3' end of the transcript and only a slight drop in efficiency for the 5' end. 10 pg shows a greater drop in efficiency and you can see more of a bias towards the 3' end. Also, comparing the level of amplification of the transcript in the library with the previous version (09272013)of the protocol (used 0.5ul final library), this protocol shows a similar level of amplification (used 0.2ul of the final library) for the 5' end of the transcript. It appears that the global level of amplification is reduced in the newer version of the protocol, hopefully this will represent fewer poly-A reads and more specific representation of transcripts. Better recovery of the 10pg RNA might be possible through reduction of RNAse III fragmentation further. Note that the curves above include 10 and 30 min pap treatments - no significant improvement in amplification was observed with the increased PAP time.


  • Combined:
    • 1203 1ng id81, 82
    • 1206 1ng id9,10, 17, 18
    • 1206 100pg id11, 12, 19, 20
    • 1206 10pg id13, 14, 21, 22
  • Ran 1ul on gel:

File:12062013 PCR1 gel2.jpg