Blue:RNA-Seq Experiments:12102013

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Revision as of 00:53, 12 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 10pg - RNase3 15s (RIII buffer)/Affymetrix PAP 10min ATP TSO.r06 T20VN_PE_R N2.id89-90 totoRNAseq
BL_ UHRR/ERCC 10pg - RNase3 30s (RIII buffer)/Affymetrix PAP 10min ATP TSO.r06 T20VN_PE_R N2.id91-92 totoRNAseq
BL_ UHRR/ERCC 10pg - RNase3 1min (RIII buffer)/Affymetrix PAP 10min ATP TSO.r06 T20VN_PE_R N2.id93-94 totoRNAseq
BL_ UHRR/ERCC 10pg - RNase3 5min (RIII buffer)/Affymetrix PAP 10min ATP TSO.r06 T20VN_PE_R N2.id95-96 totoRNAseq

Purpose

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

RNAseIII-Fragmented Library Preparation

RNA
Volume
UHRR 0.02ng/ul 0.5ul
ERCC 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-5min


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


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 15x (10pg)
72C, 5min

Results of Amplification

  • Run 5ul on gel:


File:12102013 PCR1 gel1.jpg


Results: Looks like the optimal fragmentation time is as low as 15s

Final Library Preparation

  • 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 (note: using 0.5ul of final 1ng combined library from 12062013):

File:12102013 PCR2 ERCC-3.jpg

File:12102013 PCR2 ERCC-5.jpg

File:12102013 PCR2 ERCC-30s.jpg



  • Combined id89-96 (2ul of each)
  • Ran 1ul onto gel:

File:12102013 PCR1 gel2.jpg