Blue:RNA-Seq Experiments:06042014
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Purpose[edit]
- Improve upon totoQuartzSeq protocol - optimize PCR Cylcles for 1ng UHRR
RNA-Seq Library Information[edit]
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[edit]
- 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[edit]
- 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[edit]
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[edit]
- Beads purify once (0.8x) and reconstitute in 10ul
- Run 1ul on gel:
- Quantify 1ul using Qubit:
Sample | Condition | [cDNA] ng/ul | Volume for 0.2ng/ul | Volume dH2O (final 5ul) | Nextera Barcode |
1 | RIII (11) | 0.726 | 1.38 | 3.62 | N503/N701 |
2 | RIII (11) | 0.541 | 1.85 | 3.15 | N503/N702 |
3 | RIII (13) | 1.14 | 0.88 | 4.12 | N503/N703 |
4 | RIII (13) | 0.938 | 1.07 | 3.93 | N503/N704 |
5 | RIII (15) | 2.13 | 0.47 | 4.53 | N503/N705 |
6 | RIII (15) | 2.59 | 0.39 | 4.61 | N503/N706 |
7 | RIII NTC (15) | 0.41 | 2.44 | 2.56 | N503/N707 |
8 | RIII NTC (15) | 0.508 | 1.97 | 3.03 | N503/N708 |
9 | Mg (11) | 0.366 | - | - | |
10 | Mg (11) | 0.559 | - | - | |
11 | Mg (13) | 0.52 | - | - | |
12 | Mg (13) | 0.45 | - | - | |
13 | Mg (15) | 1.11 | 0.90 | 4.10 | N504/N709 |
14 | Mg (15) | 0.9 | 1.11 | 3.89 | N504/N710 |
15 | Mg NTC (15) | 0.312 | 3.21 | 1.79 | N504/N711 |
16 | Mg NTC (15) | 0.457 | 2.19 | 2.81 | N504/N712 |
Nextera Tagmentation
Volume Tagment DNA Buffer 5ul Amplification Tagment Mix 2.5ul Diluted Sample 2.5ul Total 10ul
- Vortex and spin
- Incubate 55C 5 min
- Hold 10C
- Once sample reaches 10C add 2.5ul of NT buffer in block
- Vortex and spin
Nextera Amplification
Volume Tagmented Sample 12.5ul Nextera PCR Mix 7.5ul S5xx Primer 2.5ul S7xx Primer 2.5ul 50x Sybr green 0.08ul Total 25ul
- Mix and centrifuge
72C 3 min 95C 30s 95C 10s -> 55C 30s -> 72C 60s 8x 72C, 5min
- Run 2.5ul on gel:
- Results: Can see that the reduction of the nextera cycles resulted in a lower cDNA yield compared to 03052014 using 12 cycles - hopefully this will result in fewer clonal arrays in the final libraries
- Combined 5ul each (10ul for 9 and 10)
- beads purified twice (0.8x volume)
- Resuspended in 10ul, ran 1ul on gel: