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Old 07-07-2015, 11:16 PM   #21
cheesy
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Default 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
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Old 07-11-2015, 10:45 PM   #22
qr1120102445
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Quote:
Originally Posted by Wonghe View Post
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.
Attachment 3499
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.
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Old 07-20-2015, 08:14 AM   #23
henkvw
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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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Old 08-26-2015, 11:58 AM   #24
shamimbd7
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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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Old 08-26-2015, 05:43 PM   #25
Wonghe
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Quote:
Originally Posted by qr1120102445 View Post
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.
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Old 08-28-2015, 02:10 PM   #26
shamimbd7
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Quote:
Originally Posted by Wonghe View Post
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.
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Old 09-04-2015, 01:54 AM   #27
annrose
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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...
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Old 10-01-2015, 08:22 AM   #28
ATACJHU
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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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Old 10-01-2015, 12:49 PM   #29
jenna2790
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Quote:
Originally Posted by Wonghe View Post
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?
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Old 10-06-2015, 06:40 AM   #30
shamimbd7
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Originally Posted by annrose View Post
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.
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Old 10-19-2015, 02:17 AM   #31
Alpaca
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Hi,
Did you end-up sequencing either of these libraries shown in the bioanalyser traces? Did you get good results?
We have libraries with a relatively large peak at around 55bp (we think its primer, and it's as big as the 200bp peak) and wondered if the presence of this would ruin a sequencing run. Does anyone have any insights to offer, please? We have low sample amount so need to avoid another clean-up step if we can, but don't want to sequence rubbish either :-).

Thanks!

Kylie
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Old 10-19-2015, 03:07 AM   #32
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Quote:
Originally Posted by Alpaca View Post
Hi,
Did you end-up sequencing either of these libraries shown in the bioanalyser traces? Did you get good results?
We have libraries with a relatively large peak at around 55bp (we think its primer, and it's as big as the 200bp peak) and wondered if the presence of this would ruin a sequencing run. Does anyone have any insights to offer, please? We have low sample amount so need to avoid another clean-up step if we can, but don't want to sequence rubbish either :-).

Thanks!

Kylie
I have sent some of them out this morning, I don't know when I get my results, but I will post the bio-analyser traces and some sequencing QC when I get it back.
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Old 10-19-2015, 03:12 AM   #33
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Thanks for speedy the update, and good luck with the sequencing!

Has anyone else seen the same thing (50-70bp peak) and got good sequencing results (i.e. real sequence not sequenced-artifacts)?

Kylie
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Old 11-19-2015, 12:48 PM   #34
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Don't put too much effort to get a good bioanalyzer trace.
Bioanalyzer will give funny trace, try TBE page gel.
I know, in theory, bioanalyzer should work well, but in fact not.

Quote:
Originally Posted by shamimbd7 View Post
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.
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Old 12-16-2015, 08:19 AM   #35
Celticray
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Quote:
Originally Posted by Zaag View Post
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.
Hi Zaag,
were you be able to get a good sequencing run/result on you sample 2? It's my first time doing the ATAC-Seq and i have no clue how the Bioanalyzer result should look like.
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Old 02-22-2016, 10:21 AM   #36
mahoney
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We have been having mixed results running these samples on HS-DNA kit. Some work others seem to clog the chip. Does anyone have any suggestions on standard protocol for running these with regards to concentration. I've skipped wells....sometimes it works other times it makes no difference.
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Old 02-26-2016, 08:14 AM   #37
mantis
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Default Gel of good ATAC-seq experiment

hi everyone,

I just tried my first ATAC-seq on cells and using the protocol of Buenrostro and I obtain good results: sharp peaks, low background, (appr. 50000 cells).

I included my HS picture.

After measuring the molarity for each fragment, I obtained a good nucleosome distribution (also indicated on the gel, in the right). Before putting on the HS chip, I Ampured (1.6V) the sample.
Attached Images
File Type: jpeg ATAC-seqExample.jpeg (111.2 KB, 198 views)
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Old 02-28-2016, 10:42 PM   #38
Runuply
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Hi guys, I also used that protocol for ATAC-seq from frozen tissue, PCR amplification 5 cycles; and 7 cycles for qPCR(1 to 3rd of Max Fluorescent). It is a little hard for me to control the exact cell number, but after transposition and purification I will use 1ul to check by Qubit, which usually shows around 10ng/ul in 10ul (this should be equal to 25k cells or less). And after I did PCR amplification (in total 5+7 cycles), the purified DNA will be about 50ng/ul in 20ul.
The Bio analyzer is attached.

Last edited by Runuply; 03-02-2016 at 10:18 PM. Reason: tapping mistake
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Old 02-29-2016, 12:28 AM   #39
mantis
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Quote:
Originally Posted by Runuply View Post
Hi guys, I also used that protocol for ATAC-seq from frozen tissue, PCR amplification 5 cycles; and 7 cycles for qPCR(1 to 3rd of Max Fluorescent). It is a little hard for me to control the exact cell number, but after transposition and purification I will use 1ul to check by Qubit, which usually shows around 10ng/ul in 10l (this should be equal to 25k cells or less). And after I did PCR amplification (in total 5+7 cycles), the purified DNA will be about 50ng/ul in 20ul.
The Bio analyzer is attached.

Looks good Runuply: there seems to be a good nucleosome pattern on the gel picture. I think I would advice you to try to get rid of the primer peak at 65 before submitting the sample for sequencing.
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Old 02-29-2016, 04:37 PM   #40
Runuply
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Hi mantis, thanks for your reply. This is my 2nd test.
May I know that how do you get rid of the primer peak? Do you use something like Ampure purification method?
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