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Old 03-13-2017, 10:47 AM   #1
JDHelix
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Location: Canada

Join Date: Jan 2017
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Question A few questions about ATAC-seq library preparation. Thank you!

Hi everyone,

I've done my first ATAC-seq library preparation recently and had a few questions about the result. I would be very grateful if anyone could kindly give me some feedback/suggestions.

I used 50,000 HEK cells (fresh cells), and followed Buenrostro JD, et al. 2015 protocol exactly. After nuclei preparation -> Tn5 tagmentation (37C, 30min, on a heating block) -> PCR amplification (total cycle number was 11), I purified library with QIAGEN MinElute PCR Purification Kit and eluted in 20ul elution buffer provided by the kit. I loaded 10ul, 5ul, and 2.5ul samples on a 2% agarose gel (containing GelRed) to visualize the bands. Please see attached image.

I can see a nucleosome-like pattern at expected sizes, but not very strong. I'm wondering:
1. Is this result good enough for sequencing? Should I further optimize the condition (e.g. titrate Tn5 enzyme)?
2. There is a strong band below 100 bp (the lowest one), but I have no idea what it is. Could it be primer dimers? I have purified the library before running the gel, so I didn't expect to see primer dimers...
3. I expected to see strong signals below 150 bp, which come from sub-nucleosomal DNAs. However, I hardly see anything between 150 bp and the lowest band (indicated by a red square bracket in the image), even no background smear. Does anyone have similar experience and know why?

Thank you so much! Any comments would be highly appreciated!

Best,
Dan
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Old 03-22-2017, 08:27 AM   #2
achouak
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Location: baltimore

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hi,
i have a problem like you for atac seq. i am working in the same condition as the paper 2015 but i can not find the best results. i have many variations.
for some samples (fresh cells from mice) i can find many bands after run the gel but many others i can not i just find a large bands >1kb.
i tested 50000, 100000 and 300000 cells. i think it is better with 50000.
would you please tell what steps i have to change or to adapt to have more best results.
change our idea to ameliorate our results.
Achouak
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Old 03-22-2017, 10:04 AM   #3
JDHelix
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Location: Canada

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Hi achouak,

I'm also a new user for ATAC-seq experiment.
Usually, ATAC-seq needs 500 - 50,000 cells. I think you probably have used too many cells in some cases. According to the paper, using too many cells causes underdigestion and creates high-molecular-weight fragments. It also makes sense that 50,000 cells worked better in your hand. If I were you, I would try fewer cells.
Good luck!

Best,
Dan
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