Blue:RNA-Seq Experiments:totoRNAseq optimizations: Difference between revisions

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== Experimental Overview ==
== Experimental Overview ==


[[File:TotoRNAseq expanded optimization.jpg]]
[[File:TotoRNAseq expanded optimization.jpg|600px]]


== Purpose ==
== Purpose ==

Revision as of 01:36, 11 February 2014

Experimental Overview

File:TotoRNAseq expanded optimization.jpg

Purpose

  • Systematic analysis of different methods for addition of forward ILMN sequencing adapter to totoRNAseq libraries


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_totoRNAseq_1ngUHRR_ UHRR/ERCC 4x1ng - Mg Frag/Enzymatics PAP 5 min - STRT-Seq TSO.r06 T20VN_r01 N2.id1 totoRNAseq
UHRR/ERCC 4x1ng - RNase3 Frag/Affymetrix PAP 10 min - STRT-Seq TSO.r06 T20VN_r01 N2.id2 totoRNAseq
UHRR/ERCC 4x1ng - Mg Frag/Enzymatics PAP 5 min - Quartz-Seq xxx T20VN_r01 N2.id3 totoRNAseq
UHRR/ERCC 4x1ng - Mg Frag/Affymetrix PAP 10 min - Quartz-Seq xxx T20VN_r01 N2.id4-7 totoRNAseq
UHRR/ERCC 4x1ng - Mg Frag/Enzymatics PAP 5 min - Cel-Seq xxx T20VN_r01 N2.id3 totoRNAseq
UHRR/ERCC 4x1ng - Mg Frag/Affymetrix PAP 10 min - Cel-Seq xxx T20VN_r01 N2.id4-7 totoRNAseq
UHRR/ERCC 4x1ng - Mg Frag/Enzymatics PAP 5 min - Nugen xxx T20VN_r01 N2.id3 totoRNAseq
UHRR/ERCC 4x1ng - Mg Frag/Affymetrix PAP 10 min - Nugen xxx T20VN_r01 N2.id4-7 totoRNAseq


Primer sequences [1]


RNAseIII-Fragmented Library Preparation

RNA
Volume
2ng/ul 0.5ul
1:104 ERCC 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 min
Add 1ul of 0.1uM T20VN.id


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, combine wells
  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 ~8min to 3ul


STRT-Seq Library Preparation

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)
72C, 5min


  • Run 5ul on gel:



Results: