Andrew:Notebook/AR150602 Tn5Titrate C1
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Tn5 titration on C1 using GM12878 and mouse gDNA spike-in[edit]
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
Goal
- Compare a range of Tn5/template ratios for Tn5-059 supermutant versus EZ-Tn5
- Perform library prep directly on single cells
- Lyse with hypotonic condition and accessibilize DNA with Qiagen Protease
- Evaluate conditions based on library complexity and coverage bias