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

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Results:
Results:
== Quartz-Seq Library Preparation ==
::{| {{table}}
|-
|||||||||||<u>Volume</u>
|-
|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''

Revision as of 22:59, 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
  • Comparison of Mg-based and RNaseIII based fragmentation methods
  • Comparison of Enzymatics and Affymetrix PAP

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_id1-4 N2.id1 totoRNAseq
UHRR/ERCC 4x100pg - Mg Frag/Enzymatics PAP 5 min - STRT-Seq TSO.r06 T20VN_id5-8 N2.id2 totoRNAseq
UHRR/ERCC 10x10pg - Mg Frag/Enzymatics PAP 5 min - STRT-Seq TSO.r06 T20VN_id9-18 N2.id3 totoRNAseq
UHRR/ERCC 4x1ng - RNase3 Frag/Affymetrix PAP 10 min - STRT-Seq TSO.r06 T20VN_id1-4 N2.id4 totoRNAseq
UHRR/ERCC 4x100pg - RNase3 Frag/Affymetrix PAP 10 min - STRT-Seq TSO.r06 T20VN_id5-8 N2.id5 totoRNAseq
UHRR/ERCC 10x10pg - RNase3 Frag/Affymetrix PAP 10 min - STRT-Seq TSO.r06 T20VN_id9-18 N2.id6 totoRNAseq
UHRR/ERCC 4x1ng - Mg Frag/Enzymatics PAP 5 min - Quartz-Seq xxx T20VN_id1-4 N2.id7 totoRNAseq
UHRR/ERCC 4x100pg - Mg Frag/Enzymatics PAP 5 min - Quartz-Seq xxx T20VN_id5-8 N2.id8 totoRNAseq
UHRR/ERCC 10x10pg - Mg Frag/Enzymatics PAP 5 min - Quartz-Seq xxx T20VN_id9-18 N2.id9 totoRNAseq
UHRR/ERCC 4x1ng - RNase3 Frag/Affymetrix PAP 10 min - Quartz-Seq xxx T20VN_id1-4 N2.id10 totoRNAseq
UHRR/ERCC 4x100pg - RNase3 Frag/Affymetrix PAP 10 min - Quartz-Seq xxx T20VN_id5-8 N2.id11 totoRNAseq
UHRR/ERCC 10x10pg - RNase3 Frag/Affymetrix PAP 10 min - Quartz-Seq xxx T20VN_id9-18 N2.id12 totoRNAseq
UHRR/ERCC 4x1ng - Mg Frag/Enzymatics PAP 5 min - Cel-Seq xxx T20VN_id1-4 N2.id13 totoRNAseq
UHRR/ERCC 4x100pg - Mg Frag/Enzymatics PAP 5 min - Cel-Seq xxx T20VN_id5-8 N2.id14 totoRNAseq
UHRR/ERCC 10x10pg - Mg Frag/Enzymatics PAP 5 min - Cel-Seq xxx T20VN_id9-18 N2.id15 totoRNAseq
UHRR/ERCC 4x1ng - RNase3 Frag/Affymetrix PAP 10 min - Cel-Seq xxx T20VN_id1-4 N2.id16 totoRNAseq
UHRR/ERCC 4x100pg - RNase3 Frag/Affymetrix PAP 10 min - Cel-Seq xxx T20VN_id5-8 N2.id17 totoRNAseq
UHRR/ERCC 10x10pg - RNase3 Frag/Affymetrix PAP 10 min - Cel-Seq xxx T20VN_id9-18 N2.id18 totoRNAseq
UHRR/ERCC 4x1ng - Mg Frag/Enzymatics PAP 5 min - Nugen xxx T20VN_id1-4 N2.id19 totoRNAseq
UHRR/ERCC 4x100pg - Mg Frag/Enzymatics PAP 5 min - Nugen xxx T20VN_id5-8 N2.id20 totoRNAseq
UHRR/ERCC 10x10pg - Mg Frag/Enzymatics PAP 5 min - Nugen xxx T20VN_id9-18 N2.id21 totoRNAseq
UHRR/ERCC 4x1ng - RNase3 Frag/Affymetrix PAP 10 min - Nugen xxx T20VN_id1-4 N2.id22 totoRNAseq
UHRR/ERCC 4x100pg - RNase3 Frag/Affymetrix PAP 10 min - Nugen xxx T20VN_id5-8 N2.id23 totoRNAseq
UHRR/ERCC 10x10pg - RNase3 Frag/Affymetrix PAP 10 min - Nugen xxx T20VN_id9-18 N2.id24 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
0.5U/ul Enzymatics PAP (1:10d in H2O) 0.5ul + 0.3ul dH2O
or 600U/ul Affymetrix PAP 0.2ul + 0.6ul dH2O
40U/ul Rnase Inhibitor 0.2ul
Total 4ul


Incubate @ 37C 5 min (Enzymatics) or 4-10 min (Affymetrix)
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


Library Preparation (Mg-based Fragmentation)

RNA FRAGMENTATION
Volume
2ng/ul UHRR RNA 0.5ul
1:104 ERCC 0.2ul
5x RT buffer 1ul
dH2O 0.3ul
Total 2ul


Incubate @ 94C 2 min
Transfer to ice


End Repair
Volume
fRNA 2ul
1mM ATP 0.4ul
100mM DTT 0.1ul
RNase Inhibitor 0.1ul
PNK 0.5ul
Total 3.1ul


Incubate @ 37C 30 min


3' Tailing
Volume
PNK_fRNA 3.1ul
1mM ATP 0.5ul
0.5U/ul Enzymatics PAP (1:10d in H2O) 0.5ul
or 600U/ul Affymetrix PAP 0.2ul + 0.3ul dH2O


Incubate @ 37C 5 min (Enzymatics) or 10 min (Affymetrix)
Transfer to Ice
Add 1ul of 0.1uM T20VN.id


Reverse Transcription
Volume
PAP RNA 5ul
5xRT Buffer 1ul
10mM dNTP Mix 1ul
100mM DTT 0.25ul
Betaine (5M) 2ul
Smarter MMLV (200U/ul) 0.25ul
dH2O 0.5ul
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:

Quartz-Seq Library Preparation

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