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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 Tn5-059 to 5 ng purified DNA, which is 400 fmol/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 a minimum of 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==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Capture''' | align="center" style="background:#f0f0f0;"|'''Patient''' | align="center" style="background:#f0f0f0;"|'''1 rxn (uL)''' |- | Cell mix||Nuclei||5 |- | ||C1 suspension reagent||3.333333333 |- | |} ------------------------------------------------------------ {| {{table}} | align="center" style="background:#f0f0f0;"|'''Chamber 1''' | align="center" style="background:#f0f0f0;"|'''Reagent''' | align="center" style="background:#f0f0f0;"|'''[Initial]''' | align="center" style="background:#f0f0f0;"|'''[Final]''' | align="center" style="background:#f0f0f0;"|'''Adjust''' | align="center" style="background:#f0f0f0;"|'''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 |- | |} ------------------------------------------------------------ {| {{table}} | align="center" style="background:#f0f0f0;"|'''Tn5 dilution''' | align="center" style="background:#f0f0f0;"|'''Stock = 1X''' | align="center" style="background:#f0f0f0;"|'''Adjust -->''' | align="center" style="background:#f0f0f0;"|'''1.75''' | align="center" style="background:#f0f0f0;"|'''''' |- | 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 ------------------------------------------------------------ {| {{table}} | align="center" style="background:#f0f0f0;"|'''Chamber 4''' | align="center" style="background:#f0f0f0;"|'''Reagent''' | align="center" style="background:#f0f0f0;"|'''[Initial]''' | align="center" style="background:#f0f0f0;"|'''[Final]''' | align="center" style="background:#f0f0f0;"|'''Adjust''' | align="center" style="background:#f0f0f0;"|'''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 |- | |} ------------------------------------------------------------ {| {{table}} | align="center" style="background:#f0f0f0;"|'''Chamber 5''' | align="center" style="background:#f0f0f0;"|'''Reagent''' | align="center" style="background:#f0f0f0;"|'''[Initial]''' | align="center" style="background:#f0f0f0;"|'''[Final]''' | align="center" style="background:#f0f0f0;"|'''Adjust''' | align="center" style="background:#f0f0f0;"|'''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|600px]] [[File:Protocol Worksheet Tn5059_Titration v1-page-002.jpg|600px]] [[File:Protocol Worksheet Tn5059_Titration v1-page-003.jpg|600px]] ===Gels=== [[File:C1_OpenApp_150429_Slide1.jpg|600px]] [[File:C1_OpenApp_150429_Slide2.jpg|600px]] [[File:C1_OpenApp_150429_Slide3.jpg|600px]] [[File:C1_OpenApp_150429.pptx]]
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