activity of unloaded/unassembled Tn5
2
0
Entering edit mode
6.3 years ago

Maybe this is a dump question but will Tn5 fragment DNA into small pieces if they don't load with adapters?

next-gen-sequencing • 2.6k views
ADD COMMENT
3
Entering edit mode
15 months ago
callumjcparr ▴ 90

So I hope someone is still interested in this.

We have taken to preparing recombinant Tn5 now as it's much cheaper than commercial suppliers, whether that is unloaded or loaded. The latest batch which was particularly highly concentrated around 1.5 mg/ml led to very strong activity so much so we had to dilute the Tn5 protein 1/3 to 1/6 before loading with either Nextera or our own custom adapters. I worried there might be some nuclease carry-over from the bacterial expression system to explain why it's so much stronger than previous productions, so I included an unloaded Tn5 at the same dilution as a negative control. Well turns out this doesn't work well as a negative control because even unloaded Tn5 fragmented test lambda DNA input. Surprisingly this was even stronger than Tn5 loaded with adapters.

I tested both old and new productions of Tn5 and Tn5 from Creative Enzymes and Diagenode (purchased both unloaded versions). I contacted Diagenode and they state that they actually test the nuclease activity of unloaded Tn5 without loading adapters. Please see the tweet.

Currently what we do is form duplexes by annealing and then bind to Tn5 in a 10uL reaction for 30 mins at RT. We then degrade any unbound duplex by exoIII treatment and then EDTA inactivation. I also worried that the EDTA may have not inactivated the exoIII going forward for the tagmentation reaction so I also checked without this treatment and we still see unloaded Tn5 digest naked Lambda DNA.

This seems to disappear when we treat permeabilized nuclei. I am struggling to know the reason but of course activity of Tn5 is weaker on real chromatin and in situ compared to naked lambda DNA as there are so many steric hindrances and the issue of entering nuclei.

ADD COMMENT
3
Entering edit mode

enter image description here

enter image description here

enter image description here

enter image description here

enter image description here

enter image description here

ADD REPLY
1
Entering edit mode
15 months ago
callumjcparr ▴ 90

enter image description here

Example of TapeStation of scaled-up reaction in 500,000 nuclei. Working on the assumption we usually add 2.5 μL of complex per 50,000 nuclei/ 50 ng naked DNA, adding 25 μL of the complex. In nuclei, we do not see any nuclease contamination as the unloaded Tn5 does not show any fragmentation. I am not sure what the difference is between naked DNA and in nuclei phenomena.

ADD COMMENT
0
Entering edit mode

Appreciate your efforts but this is all off-topic here. Suggest to put this in a GitHub Pages or similar location to make it accessable for the community.

ADD REPLY
2
Entering edit mode

This is exactly related and on topic to the question asked. I am a little unsure what you mean by off-topic. The question is old but seeing as its still indexed in Google, people searching in future can find the question and actual data supporting the answer.

ADD REPLY
0
Entering edit mode

ATpoint is suggesting that these experimental data having to do with nuclease activity of Tn5 might be relevant to a biochemistry forum rather than a bioinformatics one.

However, as a consumer of both bulk and single-cell assays using Tn5 or pA-Tn5, who is occasionally asked for recommendations of experimental design, I find this to be highly relevant.

ADD REPLY
0
Entering edit mode

It is highly relevant, and off-topic in a bioinformatics forum. That's all I'm saying.

ADD REPLY
0
Entering edit mode

Ah OK. Yes both the question and answer are in the wrong forum. I will find some suitable place for this.

ADD REPLY

Login before adding your answer.

Traffic: 2579 users visited in the last hour
Help About
FAQ
Access RSS
API
Stats

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.

Powered by the version 2.3.6