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-   -   Tagmentation DNA Buffer (TD) (http://seqanswers.com/forums/showthread.php?t=41796)

dagarfield 03-20-2014 01:16 AM

Tagmentation DNA Buffer (TD)
 
Hey folks,

This seems a slim chance, but here goes: Does anyone have a recipe for the DNA Tagmentation Buffer that comes with Nextera DNA Sample Preparation Kit? We've found it helpful to wash our sample for ATAC-Seq samples with the buffer prior to Tagmentation, but the kit only comes with so much extra buffer....

bilyl 03-23-2014 03:22 AM

Try the recipe in this link?

http://www.nature.com/nprot/journal/....2013.118.html

dagarfield 03-24-2014 05:27 AM

Many thanks, this looks great. I'll update the thread with progress.

rocio 04-30-2014 01:18 AM

someone has used the recipe of this item?
Does it work well?

dagarfield 05-03-2014 09:12 AM

Works just fine. :)

bilyl 05-03-2014 03:53 PM

Quote:

Originally Posted by dagarfield (Post 139434)
Works just fine. :)

How does your ATAC-Seq data look? We're also beginning to dabble with it a bit in our lab and was wondering if you have any other optimizations.

dagarfield 05-03-2014 11:57 PM

It seems to work just fine: If you can see your post-PCR product on a gel, you'll have a fine library (in our experience). Some of the runs have more mtDNA reads than you would expect from, say, DNase-Seq, but this is something many have reported (and is seen in the data from the original paper).

In our experience, the most significant issue is simply making sure that we don't lose our pellet of cells/nuclei during the process.

I should note that we're only using the buffer recipe you linked to wash the cells. The reaction itself still takes place in the commercial buffer. Probably going to stay that way unless someone has a method for making Tn5 independent of the kit.....

rocio 05-05-2014 12:30 AM

Quote:

Originally Posted by dagarfield (Post 139434)
Works just fine. :)

Many thanks, Well try

bysanimadhu 06-04-2014 05:22 AM

hello friends,

I am planning setup ATAC-seq in our lab. Can any one send me the complete protocol.

acidcoated 07-21-2014 10:17 AM

Hi dagarfield,

What transposase are you doing the ATAC-seq with? Are you using the Nextera DNA Sample prep kit or ordering the EZ Tn5 through epicentre?

acidcoated 07-21-2014 10:27 AM

Whoops, saw that you're using the Nextera kit. I can't read apparently.
Have you tried using the Tn5 transposase through Epicentera without ordering the Nextera kit and does this work for ATAC-seq?

bjthomas 10-30-2014 08:39 AM

Quote:

Originally Posted by acidcoated (Post 145489)
Whoops, saw that you're using the Nextera kit. I can't read apparently.
Have you tried using the Tn5 transposase through Epicentera without ordering the Nextera kit and does this work for ATAC-seq?

I am also interested if anyone has used epicenters ez-tn5 to load custom oligos for ATAC-seq rather than what is found in the nextera kit.
Thanks for any input!

overclara 11-13-2014 12:16 PM

Hi all,
I'm starting with ATAC seq and would like to know if the time of incubation for Tn5 (from Nextera Kit) is relevant to have different DNA fragments in size. I was considering to do in 30 min at 37C.
thanks in advance to all

Simone78 01-05-2015 12:32 AM

Quote:

Originally Posted by overclara (Post 154653)
Hi all,
I'm starting with ATAC seq and would like to know if the time of incubation for Tn5 (from Nextera Kit) is relevant to have different DNA fragments in size. I was considering to do in 30 min at 37C.
thanks in advance to all

I guess the reason they do tagmentation at 37C and not 55C like in the Nextera protocol is just to prevent the loss (removal) of nucleosomes from the DNA. If you want to change the size distribution you have either to change the amount of transposase used (less = longer fragments and viceversa) and/or the amount of DNA.
I havent tried with the ATC protocol but if you do tagmentation with the Nextera there is no difference in the avg size of the fragments when incubating the DNA at 55C for 5, 10 or 15 mins, which probably means that the tagmentation reaction was driven to completion.

dagarfield 01-15-2015 01:39 AM

Quote:

Originally Posted by bjthomas (Post 153431)
I am also interested if anyone has used epicenters ez-tn5 to load custom oligos for ATAC-seq rather than what is found in the nextera kit.
Thanks for any input!


We've not yet tried this, but I'm considering it -- it sure would be nice to barcode during the Tagmentation step rather than the PCR step....if someone tries this, there's a beer in it for you at the next meeting.....

Simone78 01-16-2015 12:36 AM

Quote:

Originally Posted by dagarfield (Post 158123)
We've not yet tried this, but I'm considering it -- it sure would be nice to barcode during the Tagmentation step rather than the PCR step....if someone tries this, there's a beer in it for you at the next meeting.....

I am not doing ATAC-seq but I might help you anyway. Instead of spending a lot of money for few ul of Tn5 from Epicentre you could just order the plasmid from Addgene (ID: 60240) and make it yourself. Then you could all the adaptors you like!
We have recently published a paper about it, take a look!
http://www.ncbi.nlm.nih.gov/pubmed/25079858

rbd 06-18-2015 03:23 PM

ATACseq Buffer
 
Does anybody know what the MgCl2 does in the tagmentation buffer? Does it impact chromatin accessibility during ATACseq?

nucacidhunter 06-18-2015 04:29 PM

I would suggest to look at post #8 link in following thread:
http://seqanswers.com/forums/showthread.php?t=42518

intregen 07-07-2015 12:14 PM

Quote:

Originally Posted by dagarfield (Post 139458)
It seems to work just fine: If you can see your post-PCR product on a gel, you'll have a fine library (in our experience). Some of the runs have more mtDNA reads than you would expect from, say, DNase-Seq, but this is something many have reported (and is seen in the data from the original paper).

In our experience, the most significant issue is simply making sure that we don't lose our pellet of cells/nuclei during the process.

I should note that we're only using the buffer recipe you linked to wash the cells. The reaction itself still takes place in the commercial buffer. Probably going to stay that way unless someone has a method for making Tn5 independent of the kit.....

Has anyone used the homemade TD buffer for the ATAC reaction and not just for washes? Does it work just as well?

kchbio 01-04-2016 07:25 AM

Quote:

Originally Posted by intregen (Post 177067)
Has anyone used the homemade TD buffer for the ATAC reaction and not just for washes? Does it work just as well?

Nop, the home-made one does not work as efficient as the TD buffer from Illumina


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