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==Tn5 titration on the C1 using GM12878== * This is essentially a repeat of AR150602, in which the mouse DNA spike-in was not diluted 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 ===Protocol=== [[File:Protocol Worksheet AR150527_Tn5_titration v1-page-001.jpg|600px]] [[File:Protocol Worksheet AR150527_Tn5_titration v1-page-002.jpg|600px]] [[File:Protocol Worksheet AR150527_Tn5_titration v1-page-003.jpg|600px]] [[File:tempProfile.png|800px]] ====Reagents==== [[File:AR150605_reagents-page-001.jpg|600px]] [[File:AR150605_reagents-page-002.jpg|600px]] ===Results=== ====Gels==== [[File:AR150605_qPCR.png|600px]] [[File:AR150605_gelsSlide1.jpg|600px]] [[File:AR150605_gelsSlide2.jpg|600px]] ===Sequencing=== * Both fractions, 100-200 and 200-800 bp, were quantified by Qubit and qPCR. The 100-200 bp fraction had ~16 times higher concentration than the 200-800 bp, which corresponds to the gel intensity. * This indicates that the P5 and P7 regions are intact, although the smaller size fraction is too small to have actual inserts. * The 200-800 bp fraction was run on a MiSeq. The flow cell clustered at 935k/mm^2, but there were no clusters that passed filter. * The cycle intensities were extremely erratic. * If the machine was able to quantify cluster density, then the first base must have been incorporated. * It does not make sense why the sequencing would have failed. ===Follow-up=== * It is possible that the presence of the short transposon adapter sequences in the harvested amplicons are leading to off-target products during the off-chip 2nd PCR. * Cleaning up the harvested amplicons before running off-chip 2nd PCR could address this issue. * I selected 8 single-cell libraries and did Ampure XP bead purification, then repeated the off-chip PCR.
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