Blue:RNA-Seq Experiments:06042014: Difference between revisions
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*Run 1ul on gel: | *Run 1ul on gel: | ||
[[File:06052014_PCR1_Gel1.jpg|500px]] | |||
*Quantify 1ul using Qubit: | *Quantify 1ul using Qubit: |
Revision as of 18:27, 9 June 2014
Purpose
- Improve upon totoQuartzSeq protocol - optimize PCR Cylcles for 1ng UHRR
RNA-Seq Library Information
Library ID | Sample | Condition | 5' Adaptor | 3' Adaptor | N2 PCR Rev Primer | RNA-Seq Protocol |
2x1ng UHRR | RIII Frag - 11 PCR cycles | Quartz_Tagging | Quartz-RT | Quartz-Suppression | totoRNAseq/Quartz-Seq | |
2x1ng UHRR | RIII Frag - 13 PCR cycles | Quartz_Tagging | Quartz-RT | Quartz-Suppression | totoRNAseq/Quartz-Seq | |
2x1ng UHRR | RIII Frag - 15 PCR cycles | Quartz_Tagging | Quartz-RT | Quartz-Suppression | totoRNAseq/Quartz-Seq | |
NTC | RIII Frag - 15 PCR cycles | Quartz_Tagging | Quartz-RT | Quartz-Suppression | totoRNAseq/Quartz-Seq | |
2x1ng UHRR | Mg Frag - 11 PCR cycles | Quartz_Tagging | Quartz-RT | Quartz-Suppression | totoRNAseq/Quartz-Seq | |
2x1ng UHRR | Mg Frag - 13 PCR cycles | Quartz_Tagging | Quartz-RT | Quartz-Suppression | totoRNAseq/Quartz-Seq | |
2x1ng UHRR | Mg Frag - 15 PCR cycles | Quartz_Tagging | Quartz-RT | Quartz-Suppression | totoRNAseq/Quartz-Seq | |
NTC | Mg Frag - 15 PCR cycles | Quartz_Tagging | Quartz-RT | Quartz-Suppression | totoRNAseq/Quartz-Seq |
Primer sequences [1]
cDNA Preparation: RNAseIII-Based Fragmentation
- RNA
Volume 2ng/ul UHRR 0.5ul 1:10e4 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
- Add 25ul beads per well
- Allow to bind 15 min
- Remove supernatant and wash 2x with 1ml 80% EtOH
- Air Dry 10min or until cracking first occurs
- Resuspend beads in 6ul ExoI reaction (see below):
cDNA Preparation: Mg-based Fragmentation
- RNA FRAGMENTATION
Volume 2ng/ul UHRR 0.5ul 1:10e4 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 40U/ul 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 600U/ul Affymetrix PAP 0.2ul dH2O 0.2ul Total 4
- Incubate @ 37C 10 min
- Transfer to Ice
- Add 1ul of 0.1uM Quartz-RT [Quartz-Seq]
- Reverse Transcription
Volume PAP RNA 5ul 5xRT Buffer 1ul 10mM dNTP Mix 1ul 100mM DTT 0.25ul Betaine (5M) 2ul 40U/ul Rnase Inhibitor 0.1ul Smarter MMLV (200U/ul) 0.25ul dH2O 0.4ul Total 10ul
- Incubate @ 42C 60 min
- Beads Purification
- Add 25ul beads per well
- Allow to bind 15 min
- Remove supernatant and wash 2x with 1ml 80% EtOH
- Air Dry 10min or until cracking first occurs
- Resuspend beads in 6ul ExoI reaction (below):
Quartz-Seq Library Preparation
RT Primer Removal
Volume 10x PCR Buffer (homemade Takara) 0.4ul 10x Exo I buffer (epicentre) 0.2ul 100mM DTT 0.2ul Exonuclease I (epicentre) 0.6ul dH2O 4.6ul 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 (ambion) 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 12-15x (1ng) 68C, 5min
Library Quantification and Nextera Preparation
- Beads purify once (0.8x) and reconstitute in 10ul
- Run 1ul on gel:
- Quantify 1ul using Qubit: