ATAC-seq protocol stop-point check
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4 months ago
Lord.Mor • 0

Hi everyone! I'm performing ATAC-seq to determine open chromatin regions of a cell line. I went through the protocol and wonder if there are means to verify during the whole procedure if we are getting a result or even a glimpse of the result? Like a stop-point and see a preview of the end-product. Is there such a way? or the closest thing is performing library-quality through bioanalyzer after PCR amplification? Thank you very much in advance.

Oh, there is actually a stopping point before PCR amplification, wherein we can store the purified DNA at -20 after tagmentation and before PCR amplification. Is there a way or possibility that at this stop point, we can have a preview of the result or check whether we are doing the right thing? Thanks once again. Keep safe!

ATAC-seq • 495 views
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4 months ago
ATpoint 54k

Is there a way or possibility that at this stop point, we can have a preview of the result or check whether we are doing the right thing?

No, the Bioanalyzer track is the only proxy you can get and even this does not necessarily mean that the data are good. You must see the banding pattern though, otherwise something went wrong and it would imho make little sense to sequence it.

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It's clear now. I'm just curious that is why I asked the question. Thank you very much, ATpoint, for the response. Keep safe!

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Sure thing. I know by experience that it is tempting to know early on whether the experiment worked fine, unfortunately with NGS this is not possible most of the time. A shallow sequencing is sometimes a good idea, e.g. for ChIP-seq where success is not guaranteed and strongly dependent on factors such as antibody and sonication/IP conditions. ATAC-seq (in my hands for human and mouse) kind of always works. In the end only the sequencing itself tells you success/failure.

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Yeah, it is really tempting. I guess I just have to really wait for the sequencing then. Big thanks again.

Oh! I have other questions but not related to the previous one.

  1. May I do a paired-end 150 bp (2x150bp) sequencing? Or do you recommend other read lengths?
  2. Please correct me if I'm wrong. These 2x150 bp fragment sequences will be further trimmed down to remove adapters before genome alignment, right? I have seen articles where they did 2x50bp, 2x100bp, and 2x150bp. That is why I'm confused if I should do 2x150bp, 2x100bp, or 2x50bp.

P.S. I actually saw your response to one of the questions asked on the link: ATAC-seq 150bp reads .

Thank you!

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Do what is cheapest. If you have agood quote for a Novaseq run where 2x150 is often standard then do this, or if 2x50 is cheaper then go for this one. We usually do 2x50bp.

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Big, big thanks ATpoint. Keep safe.

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