Blue:RNA-Seq Experiments:01082014

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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_10pgUHRR_Jan08 UHRR/ERCC 10pg - RNase3 15s/Affymetrix PAP 1min TSO.r06 T20VN_PE_R N2.id1-2 totoRNAseq
UHRR/ERCC 10pg - RNase3 15s/Affymetrix PAP 2min TSO.r06 T20VN_PE_R N2.id3-4 totoRNAseq
UHRR/ERCC 10pg - RNase3 15s/Affymetrix PAP 4min TSO.r06 T20VN_PE_R N2.id5-6 totoRNAseq
UHRR/ERCC 10pg - RNase3 15s/Affymetrix PAP 6min TSO.r06 T20VN_PE_R N2.id7-8 totoRNAseq
UHRR/ERCC 10pg - RNase3 15s/Affymetrix PAP 8min TSO.r06 T20VN_PE_R N2.id9-10 totoRNAseq
UHRR/ERCC 10pg - RNase3 15s/Affymetrix PAP 10min TSO.r06 T20VN_PE_R N2.id11-12 totoRNAseq
UHRR/ERCC 1ng - RNase3 1min/Affymetrix PAP 10min TSO.r06 T20VN_PE_R N2.id13-14 totoRNAseq


Primer sequences [1]

Purpose[edit]

  • Test optimal PAP treatment time for 10pg RNA based on 12062013
  • Use ERCCs to determine level of transcript amplification between libraries


RNAseIII-Fragmented Library Preparation[edit]

RNA
Volume
UHRR 2ng/ul or 0.02ng/ul 0.5ul
ERCC 1:10E4 or 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 15s-1min


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 1-10 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); 15x (10pg)
72C, 5min

Results of Amplification[edit]

  • Run 5ul on gel:

File:01082013 PCR1 Gel1.jpg


Results: Global amplification levels seem a little low, but otherwise looks fine. Some variability with the 10pg that could be due to the beads purification stage following RT...

Final Library Preparation[edit]

  • Beads purified twice at (0.8x), re-suspended in 10ul
  • PCR on 0.5ul each library using ILMN_PCR_F and either ERCC130R227 or ERCC130R964.

File:01082014 ERCC130 R227.jpgFile:01082014 ERCC130 R964b.jpg

Results:

  • Still variable amplification levels seen with 10pg. More consistency might be achieved by combining 10pg samples for beads purification prior to TSO/PCR
  • Can see at least one of the 2min or 4 min showing similar levels as the 1ng (especially for the R964 - i.e.3'end) while longer PAP times tended to show poorer amplification curves... therefore, 4 min PAP may be sufficient for 10pg RNA quantities
  • Its difficult to fully conclude optimal conditions due to the variability... hopefully this is an artifact of the single 10pg beads purification rather than inconsistency/inefficiency of the totoRNAseq reactions at these minimal RNA concentrations