Is my ATACseq library okay?

coralxanpu · 05-21-2018, 04:28 PM

We are new to ATAC-Seq. We have performed library validation and several of our samples have produced results similar to those in the image attached. We have a single large peak at 186 bp. Are we okay to go ahead for sequencing? Thanks a lot.

coralxanpu · 05-21-2018, 05:19 PM

I forgot to mention that above samples are from FACS mouse cells. We also tried IP instead of FACS, which gave us multiple small peaks, which method shall I go on? Any suggestion is greatly appreciated.

pmiguel · 05-23-2018, 12:00 PM

We run qc chips for people doing ATACseq with some frequency. I'm never sure what a "good" result is.
I guess you want a large percentage of your DNA to be in the nucleosome monomer size range? But you also want to see some multimers? Not sure.
--
Phillip

Affef · 05-30-2018, 07:34 AM

It seems that your library is over tagmented. Maybe you should decrease the incubation time while performing the tagmentation.

Simone78 · 06-07-2018, 05:09 AM

I would also say that it is over-tagmented. Which Tn5 are you using, Nextera or home-made? Maybe there is too much Tn5 for the number of cells you are using?

RickC7 · 06-14-2018, 09:17 AM

Here is what my ATAC libraries look like. I agree that optimization of tagmentation enzyme amount or time should be considered.

What is you input amount? Are you starting with <50,000 cells? Smaller input amounts will require less enzyme and/or shorter incubation.

Grg91 · 12-11-2018, 01:01 AM

Did you solve this? My libraries look like yours

María Camila Fetiva · 03-01-2019, 06:55 AM

Hello, I performed ATACseq preparation of librarys I used 50.000 cells counted by en bauer chamber, and following the protocol of Buen rostro, I did 25 cycles in total of PCR. Can someone tell me if this library looks ok? (attached pdf 1st sample) I am not sure about it. Thank you so much for your help

Best

Camila

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12-11-2018, 01:01 AM	#7
Grg91 Junior Member Location: switzerland Join Date: Dec 2018 Posts: 5	same profile Did you solve this? My libraries look like yours

05-21-2018, 05:19 PM	#2
coralxanpu Junior Member Location: LA Join Date: May 2018 Posts: 3	I forgot to mention that above samples are from FACS mouse cells. We also tried IP instead of FACS, which gave us multiple small peaks, which method shall I go on? Any suggestion is greatly appreciated.

05-23-2018, 12:00 PM	#3
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	We run qc chips for people doing ATACseq with some frequency. I'm never sure what a "good" result is. I guess you want a large percentage of your DNA to be in the nucleosome monomer size range? But you also want to see some multimers? Not sure. -- Phillip

05-30-2018, 07:34 AM	#4
Affef Junior Member Location: Germany Join Date: Mar 2018 Posts: 1	It seems that your library is over tagmented. Maybe you should decrease the incubation time while performing the tagmentation.

06-07-2018, 05:09 AM	#5
Simone78 Senior Member Location: Basel (Switzerland) Join Date: Oct 2010 Posts: 208	I would also say that it is over-tagmented. Which Tn5 are you using, Nextera or home-made? Maybe there is too much Tn5 for the number of cells you are using?