[rbd]

Has anybody tried increasing the cell lysis time?

[nsmackler]

If this works, does that mean that both the plasma membrane and the nuclear envelope are permeable to the transposase?

It means that enough of the cells are osmotically lysed in the TD buffer.

[Wonghe]

frozen tissue

Hi All, I've been trying to do ATAC-seq on nuclei isolated from frozen tissues with sucrose gradient. Although I am able to obtain a nucleosome-like pattern (see above attachment) but after sequencing I get many background peaks which are very noisy and the peaks at the TSS were not distinct. Anyone has any idea to improve the method and reduce the background peaks?

[ksingh]

I am looking for methods for alternate to CHIP-Seq for Mouse hematopoietic stem cells. I can't get large number of cells for this protocol. I would appreciate help, if any body aware of protocol (reference). Thanks. KPS

[Zaag]

Hi wen yuan,
Attached is what I got from the authors, or someone from their lab. I'm also waiting for permission to their forum, let's see...
Hope it helps

I used this protocol and got this after adapter-PCR:





Can anyone comment? Is it completely crap or not?

To me sample 2 looks better than sample 1, but I have no real clue as in how it should look.

[cheesy]

ATACseq bioanalyser trace

Hi

Can someone who has been successful with ATACseq please post a bioanalyser trace of their results? Im interested to see what the expected result should look like.

Thanks

[qr1120102445]

A correction to my previous post, tagmentation was done at 37C for 30min in the below conditions:
1) 20K cells, 10ul Tag reaction vol (1ul enzyme) no clean up before PCR (10cycles) clean up using 1:1 Ampure beads and ran on Bioanalyzer (~200-400bp)

2) 20K cells, 25ul Tag reaction vol (2ul enzyme) clean up in Qiagen MinElute col elute in 10ul and use all the transpose DNA for 10 cycles PCR, ran 10ul of amplified product on a 6% TBE gel (observed smear).

So far no luck in the nucleosome pattern.
[ATTACH]3499[/ATTACH]

Did you solve your problem of no nucleosomal pattern after library now? Did you sequenced your library? I also faced the same problem. Even though tried to optimize the cell number, incubation time, still didn't work.

[henkvw]

Hi all,
Very newbie question here, I'm quite new to the whole next-generation sequencing;
I'm now generating ATAC-seq libraries from a mouse cell line. I have 8 different samples that I can amplify with different barcoded primers as to be able to multiplex during sequencing. My question is: How many reads would I need to obtain in order to acquire sufficient coverage of the mouse genome? Would I be able to pool and multiplex my 8 samples or do I need to run separate runs? I can use the MiSeq or HiSeq here for sequencing; which one would be preferable keeping in mind my questions above?

Many thanks in advance for helping me out!

[shamimbd7]

Hi all,

I am trying to do ATAC-Seq experiment using plant samples. I am wondering whether there is any size selection step after PCR amplification ( Using ampure beads). If yes, what size I should select. OR Instead of size selection, should I just clean up my PCR products using Qiagen Mini Elute.

My second question is, whether the fixation of nuclei with formaldehyde affects the library making even if I do de-crosslinking after the tagmentation reaction.

I would highly appreciate your response.

[Wonghe]

Did you solve your problem of no nucleosomal pattern after library now? Did you sequenced your library? I also faced the same problem. Even though tried to optimize the cell number, incubation time, still didn't work.

Yes I get my atac to work on 70K freshly trypsinize cells using the original Greenleaf protocol. Depending on the cell type you are working on you might need to adjust the lysis buffer content to make sure the cells are lysed before adding the transposase.

[shamimbd7]

Yes I get my atac to work on 70K freshly trypsinize cells using the original Greenleaf protocol. Depending on the cell type you are working on you might need to adjust the lysis buffer content to make sure the cells are lysed before adding the transposase.

Hi Wonghe,

I am very interested to see bioanalyzer trace of a successful ATAC-Seq experiment. If possible, would you please share your bioanalyzer trace. If not, would you please describe how was the pattern.

[annrose]

Hi all

I am trying to do ATAC-seq on fromaldehyde fixed cells. But dont have much sucess with it. Was wondering if any of you have tried it ......any suggestion will be of great help...

[ATACJHU]

Hi All

I am trying to do ATACseq, and have a question.

How to set up qPCR threshold and baseline in order to see five cycle amplification plot? The regular qPCR has default 3-15 cycles as baseline, I do not think that would be suitable for the ATAC library amplification. I am using StepOne Plus machine.

Thank you guys!

[jenna2790]

Follow-uo

Hi All, I've been trying to do ATAC-seq on nuclei isolated from frozen tissues with sucrose gradient. Although I am able to obtain a nucleosome-like pattern (see above attachment) but after sequencing I get many background peaks which are very noisy and the peaks at the TSS were not distinct. Anyone has any idea to improve the method and reduce the background peaks?

I am also using frozen tissues. Were you able to eliminate the noise?

[shamimbd7]

Hi all

I am trying to do ATAC-seq on fromaldehyde fixed cells. But dont have much sucess with it. Was wondering if any of you have tried it ......any suggestion will be of great help...

Hi Annrose, I have isolated formaldehyde fixed nuclei and thinking to do ATAC-Seq experiment on that. Would you please mention whether you are getting long fragments after tagmentation or any other issues? I am also wondering whether you have reverse crosslinked your samples before going for PCR. I would highly appreciate your response.