Andrew:Notebook/C1 OpenApp 150429
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C1 OpenApp 150429
- Tn5-059 vs. EZ-Tn5 on GM12878
Motivation
- Library complexity depends on the ratio of Tn5 to template.
- Tn5-059 concentration is normalized to EZ-Tn5. The current batch of Tn5-059 is 25X = 17.5 uM (1X = 700 nM).
- Illumina uses 40 nM Tn5-59 to 5 ng purified DNA, which is 400 fmole/ng assuming a 50 uL reaction
- I have been using 0.1X = 70 nM to 6 pg for a single cell, which is 368 fmole/ng in a 31.5 nL volume
- Each fragment requires adapters on both ends; half will be orange/orange or blue/blue, which are non-functional.
- A million unique reads will therefore consume 4 million molecules of Tn5
- 70 nM in 31.5 nL is approximately 1.33 billion molecules
Prep
Notes
- Tn5 mix must be backloaded
- Sample: GM12878
Reagents
Capture | Patient | 1 rxn (uL) |
Cell mix | Nuclei | 5 |
C1 suspension reagent | 3.333333333 | |
Chamber 1 | Reagent | [Initial] | [Final] | Adjust | 1 rxn (uL) |
HLS mix | H2O | NA | NA | NA | 4.35 |
Volume (uL) | Tris-HCl (M) | 0.4 | 0.03 | 1.5 | 0.75 |
10 | Qiagen Protease (mg/mL) | 10 | 4.40 | 1.5 | 4.40 |
C1 Loading Reagent (X) | 20 | 1.00 | 1 | 0.50 | |
Sum (uL) | 10.00 | ||||
Tn5 dilution | Stock = 1X | Adjust --> | 1.75 | ' |
Final (uL) | Final X | Tn5 (uL) | H2O (uL) | |
72 | 0.2 | 37.8 | 70.2 | of 1X mix |
72 | 0.1 | 36 | 36 | of 0.2X mix |
72 | 0.05 | 36 | 36 | of 0.1X mix |
72 | 0.025 | 36 | 36 | of 0.05X mix |
- Repeat for EZ-Tn5
- Backload 5 ul each
Chamber 4 | Reagent | [Initial] | [Final] | Adjust | 1 rxn (uL) |
ProtQ mix | H2O | NA | NA | NA | 20.41 |
Volume (uL) | Tris-HCl (M) | 0.40 | 0.03 | 1 | 1.80 |
24 | Qiagen Protease (mg/mL) | 10 | 0.20 | 1.233 | 0.59 |
C1 Loading Reagent (X) | 20 | 1.00 | 1 | 1.20 | |
Sum (uL) | 24.00 | ||||
Chamber 5 | Reagent | [Initial] | [Final] | Adjust | 1 rxn (uL) |
PCR mix | H2O | NA | NA | NA | 5.70 |
Volume (uL) | NPM (X) | 3.33 | 1.00 | 2.233 | 20.12 |
30 | Nextera Adapter 1 (uM) | 10.00 | 0.20 | 2.233 | 1.34 |
Nextera Adapter 2 (uM) | 10.00 | 0.20 | 2.233 | 1.34 | |
C1 Loading Reagent (X) | 20 | 1.00 | 1 | 1.50 | |
Sum (uL) | 30.00 | ||||
Protocol
File:Protocol Worksheet Tn5059 Titration v1-page-001.jpg
File:Protocol Worksheet Tn5059 Titration v1-page-002.jpg
File:Protocol Worksheet Tn5059 Titration v1-page-003.jpg
Gels
File:C1 OpenApp 150429 Slide1.jpg
File:C1 OpenApp 150429 Slide2.jpg