Andrew:Notebook/C1 OpenApp 150429

From ZhangLabWiki
Revision as of 20:53, 19 May 2015 by >Andrew (→‎Reagents)
Jump to navigation Jump to search

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

File:C1 OpenApp 150429 Slide3.jpg

File:C1 OpenApp 150429.pptx