Blue:RNA-Seq Experiments:06042014

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Revision as of 21:53, 3 June 2014 by >B1lake
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Purpose

  • Improve upon totoQuartzSeq protocol


RNA-Seq Library Information

Library ID Sample Condition 5' Adaptor 3' Adaptor N2 PCR Rev Primer RNA-Seq Protocol
2x1ng UHRR 2min Mg Frag/Enzymatics PAP 5 min Quartz_Tagging Quartz-RT Quartz-Suppression totoRNAseq/Quartz-Seq
2x100pg UHRR 2min Mg Frag/Enzymatics PAP 5 min Quartz_Tagging Quartz-RT Quartz-Suppression totoRNAseq/Quartz-Seq
3x10pg UHRR 2min Mg Frag/Enzymatics PAP 5 min Quartz_Tagging Quartz-RT Quartz-Suppression totoRNAseq/Quartz-Seq
NTC 2min Mg Frag/Enzymatics PAP 5 min Quartz_Tagging Quartz-RT Quartz-Suppression totoRNAseq/Quartz-Seq
2x1ng UHRR 0.1U RNase3 Frag/Affymetrix PAP 10 min Quartz_Tagging Quartz-RT Quartz-Suppression totoRNAseq/Quartz-Seq
2x100pg UHRR 0.1U RNase3 Frag/Affymetrix PAP 10 min Quartz_Tagging Quartz-RT Quartz-Suppression totoRNAseq/Quartz-Seq
3x10pg UHRR 0.05U RNase3 Frag/Affymetrix PAP 5 min Quartz_Tagging Quartz-RT Quartz-Suppression totoRNAseq/Quartz-Seq
NTC 0.05U RNase3 Frag/Affymetrix PAP 4 min Quartz_Tagging Quartz-RT Quartz-Suppression totoRNAseq/Quartz-Seq


Primer sequences [1]


cDNA Preparation: RNAseIII-Based Fragmentation

RNA
Volume
2ng/ul or 0.2ng/ul or 0.02ng/ul 0.5ul
1:10E4 or 1:10E5 or 1:10E6 ERCC 0.2ul
0.1uM Quartz-RT (see below) 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 4-10 min
Add 1ul of 0.1uM Quartz-RT [Quartz-Seq]


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 60 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 ~8min to 3ul


Quartz-Seq Library Preparation

RT Primer Removal


Volume
Purified cDNA 3ul
10x PCR Buffer 0.4ul
10x Exo I buffer 0.2ul
100mM DTT 0.2ul
Exonuclease I 0.6ul
dH2O 1.6ul
Total 6ul
Takara Taq PCR Buffer:
Volume
1M Tris.HCl (pH 8.3) 100ul (100mM Final)
1M KCl 500ul (500mM Final)
1M MgCl2 15ul (15mM Final)
dH2O 385ul
Total 1ml


Add directly to beads and mix
Incubate @37C 30min
Incubate @80C 20min
Incubate @4C


Poly-A Tailing

Volume
ExoI RXN 6ul
10x PCR Buffer 0.5ul
10mM dATP 1.5ul
RNaseH 0.12ul
TdT enzyme (Roche 03333566001) 0.42ul
dH2O 2.46ul
Total 11ul


Incubate @37C 50 sec
Incubate @65C 10min
Incubate @4C


Second Strand Synthesis


Volume
Poly-A Tailed Sample 11ul
2x Terra Direct PCR Buffer 25ul
10uM Tagging Primer 0.32ul
Terra Polymerase 2ul
dH2O 18.68ul
Total 57ul
Incubate @98C 2min 10 sec
Incubate @40C 1min
Incubate @68C 5min
Transfer to ice


PCR Amplification

Volume
2nd Strand Sample 57ul
2x Terra Direct PCR Buffer 25ul
100uM PCR Primer 1ul
dH2O 24ul
Total 107ul


68C 1 sec
98C 10s -> 65C 15s -> 68C 5min 15x (1ng); 18x (100pg); 21x (10pg)
68C, 5min


Ran 5ul onto gel: