Has anybody tried increasing the cell lysis time?
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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?Attached Files
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Originally posted by Jubs View PostHi 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
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.Last edited by Zaag; 06-30-2015, 06:01 AM.
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Originally posted by Wonghe View PostA 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]
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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!
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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.
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Originally posted by qr1120102445 View PostDid 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.
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Originally posted by Wonghe View PostYes 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.
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.
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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!
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Follow-uo
Originally posted by Wonghe View PostHi 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?
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Originally posted by annrose View PostHi 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...
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