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Old 11-29-2018, 05:27 AM   #1
lapensee
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Default Inefficient ATAC-seq library prep amplification

Hi all,

I’m having trouble generating an “optimal” library for sequencing.
I am using a human cell line derived from an adrenocortical tumor (from ATCC).

I have tried 12.5, 25, 50, and 100k cells with 2.5ul Tn5 (Illumina).
I've tried lysing with and without Digitonin, no difference.

qPCR determined that I needed ~5-7 extra cycles of amplification.

Qiagen cleanup was not removing the primer, and I had large peaks at ~2000 or bigger, so I switched to AMPure beads (double sided purification, first 0.5X then 1.8X)

I never seem to get those nice big peaks around ~<200, 400 just a blip. See pics below.

Does anyone have any ideas for improving the efficiency of my library amplification?

Thanks
Chris
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File Type: jpg ATAC-Seq library QC.jpg (92.5 KB, 17 views)
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Old 12-03-2018, 08:41 AM   #2
lapensee
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If not suggestions, would someone be willing to post a pic of a GREAT bioanalyzer profile (for ATAC-Seq)?
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Old Yesterday, 11:15 AM   #3
lapensee
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Default Undertagmentation

Suspecting undertagmentation. Anyone else run into this problem, and if so, what was your remedy?
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File Type: jpg Undertagmentation2.jpg (89.9 KB, 4 views)

Last edited by lapensee; Yesterday at 11:25 AM. Reason: Add figure
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Old Yesterday, 04:38 PM   #4
Meyana
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I have seen the same thing repeatedly with FACS sorted nuclei isolated from frozen tissue, hoping someone has an idea on how to optimize...
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