Blue:RNA-Seq Experiments:09102013

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Revision as of 00:22, 10 September 2013 by >B1lake (→‎TSO Quantification)
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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_ERCC_N2id1_ ERCC 1ng - Fragmented TSO.r04 T20V.id1 N2.id1 totoRNAseq QC qPCR Analysis
BL_ERCC_N2id2_ ERCC 1ng - Unfragmented TSO.R04 T20V.id1 N2.id2
BL_ERCC_N2id3_ ERCC 1ng - Fragmented TSO.R05 T20V.id1 N2.id3
BL_ERCC_N2id4_ ERCC 1ng - Unfragmented TSO.R05 T20V.id1 N2.id4
BL_ERCC_N2id5_ ERCC 1ng - Fragmented Smarter TSO T20V.id1 N2.id5
BL_ERCC_N2id6_ ERCC 1ng - Unfragmented Smarter TSO T20V.id1 N2.id6
BL_ERCC_N2id7_ ERCC 1ng - Fragmented TSO.R04 Supp.T20.id1 N2.id7
BL_ERCC_N2id8_ ERCC 1ng - Unfragmented TSO.R04 Supp.T20.id1 N2.id8


Purpose

  • Examine efficiency of TSO primers using ERCC assay
  • Examine efficiency of TSO between fragmented and unfragmented using ERCC assay


Library Preparation (RNAseIII Fragmentation):

RNA FRAGMENTATION
Volume
1ng RNA 0.5ul
1:1E4 0.2ul
10x RNaseIII Buffer 0.2ul
0.1uM T20V.id1 0.1ul
dH20 0.25ul
Total 1.25ul
Incubate @ 72C 3 min
Incubate @ 37C 1min
Incubate @ 4C 10min


RNA FRAGMENTATION
Volume
RNA 1.25ul
RNase III 0.08ul
dH20 0.67ul
Total 2ul


Incubate @ 37C 10 min
Add 1.1ul dH2O
Incubate @ 65C 10 min


3' Tailing
Volume
PNK_fRNA 3.1ul
5x RT Buffer 0.5ul
1mM ATP 0.25ul
0.5U/ul PAP (1:10d in H2O) 0.25ul


Incubate @ 37C 5 min
Transfer to Ice


Reverse Transcription:

Volume
PAP RNA 4.1ul
10mM dNTP Mix 1ul
0.1uM T20V.ID 0.9ul


Incubate @ 65C 3 min
Transfer to Ice


Volume
5xRT Buffer 1.5ul
100mM DTT 0.17ul
Betaine (5M) 2ul
RNase Inhibitor (40U/ul) 0.1ul
Smarter MMLV (200U/ul) 0.23ul
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

TSO (x2)
Volume
Purified cDNA 3ul
5x RT buffer [MMLV] 2ul
10mM dNTP mix 1ul
100mM DTT 0.25ul
10uM TSO.r04 1ul
Betaine (5M) 2ul
RNase Inhibitor 0.25ul
Smarter MMLV 0.5ul
Total 10ul


Incubate @42C 10min
Incubate @70C 10min


USER
Add 1ul and incubate at 37C for 30 min


PCR (PhusionHF)
Volume
TSO Reaction 11ul
2x Buffer 25ul
10uM PB_PCR_F 1ul
10uM PCR_N2_idX 1ul
H2O 12ul
Total 50ul


98C 30 sec
98C 15s -> 58C 20s -> 72C 30s 6x
98C 10s -> 72C 20s 15x
72C, 5min


Results of Amplification

  • Ran 1ul on gel:


TSO Quantification

  • Comparison of Pre-amp and post-amp plots:
PCR - 5' TSO PCR - 3' T20 PCR fragment
TSO+130-5' T20+130-5' ERCC-130-5'
TSO+130-mid T20+130-mid ERCC-130-mid
TSO+130-3' T20+130-3' ERCC-130-3'
TSO+130-5' T20+130-5' ERCC-130-5'
TSO+130-mid T20+130-mid ERCC-130-mid
TSO+130-3' T20+130-3' ERCC-130-3'
TSO=ILMN_PCR_F; T20=ILMN_PCR_R
  • Amplified 1ul TSO and 5ul PCR using Sybr-Green:
Volume
Library 1ul or 5ul
2xSybr Buffer 25ul
10uM F primer 1ul
10uM R Primer 1ul
dH2O 22ul or 18ul
Total 50ul


95C 30sec
95C 10s -> 60C 20s -> 72C 30s => 5X
72C, 5min


  • Ran ul on gel: