Tagmentation DNA Buffer (TD)

[dagarfield]

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]

Try the recipe in this link?

http://www.nature.com/nprot/journal/v8/n10/full/nprot.2013.118.html

[dagarfield]

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

[rocio]

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

[dagarfield]

Works just fine.

[bilyl]

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]

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]

Works just fine.

Many thanks, We´ll try

[bysanimadhu]

hello friends,

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

[acidcoated]

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]

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]

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]

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 37°C.
thanks in advance to all

[Simone78]

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 37°C.
thanks in advance to all

I guess the reason they do tagmentation at 37°C and not 55°C 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 haven´t 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 55°C for 5, 10 or 15 mins, which probably means that the tagmentation reaction was driven to completion.

[dagarfield]

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.....