Anyone optimised ATAC-seq for Nextera XT?

[ScottC]

Hi there,

Has anyone tried/optimised ATAC-seq using Nextera XT?

Cheers,

Scott.

[kropak1807]

I have the same question - let me know if you figure it out. Im not sure which is better, Nextera or NexteraXT? Nextera XT kit is much cheaper, but im assuming that is because there is signigficantly less enzyme provided.

Does anyone know how many ATAC-seq samples can be run on Nextera vs Nextera XT kits?

[Julianix]

Nextera XT?

Hi, same question, does anyone had tried Nextera XT for ATACseq?

if Nextera XT is for low genomic input, I would assume that you can us it for ATACseq with LOW NUMBER of cells, but probably the results won't be comparable with Libraries made with the regular kit as those will be more tagged... I personally do my ATACseq with 10k cells, so I´m already in the low number of cells. As I`m going to start a new project I thought to use the XT kit and increase the incubation time to 45 min instead of 30. Suggestions?

I think more importantly would be to know if the barcodes introduced by XT are the same introduced by the regular kit, meaning that you can use the same barcodes to amplify the library.

[kevin199011]

I'm also very curious if anyone has optimized ATAC-seq with Nextera XT. Our lab has been using Nextera XT kit for single cell RNA-seq and it would great we can just use the same kit for ATAC-seq also.
Many thanks.

[laclotze]

For anyone who's interested, I have just compared library amplification efficiencies with Tn5 (Amplification Tagmentation Mix) from the old Nextera XT kit and Tn5 (Tagment DNA Enzyme I) that used to be supplied with the old Nextera kit.

Protocol details:
- 50,000 nuclei isolated from Arabidopsis seedlings per reaction
- 2.5 ul Tn5 per 50 ul tagmentation reaction. 37˚C for 30 mins
- Fragments cleaned up with Qiagen's MinElute kit
- NEB's Q5 used for initial library amplification (5 cycles)
- qPCR (Roche, SYBR Green I) used to estimate the number of additional cycles necessary for library amplification

The Cp values for the Nextera Tn5 and Nextera XT Tn5 were 5.55 and 12.75 respectively, so it looks like there is quite a difference in the amount of enzyme supplied in the respective kits. How much this affects the sequencing I don't know, but I might include a Nextera XT-derived sample in a future round to find out.