Fixed! Illumina is now selling the Tn5 transposase (and TD buffer) separately...

Hi. I also just came across this issue. Could you provide a link to where you can order transposase and buffer separately? I couldn't find it on the illumina website.

Hi Christoph Dotter,

We directly asked our local Illumina manager, and he confirmed that they sell it separately now. Furthermore, he sent us a purchase quotation, which I am not allowed to spread. Anyway, this is a usual Tn5 (15027865) from the "old" kit. Probably, you just need to contact respective Illumina person in Austria.

Best,

Michail

Hi Michail Yekelchyk,

thanks for the answer and for providing the catalogue number of the transposase. I'll contact Illumina and ask for a quote.

Best,

Christoph

I'm trying to use the Nextera DNA flex library prep kit to perform ATAC-seq. Could you tell me why bead bound transposase is not suitable for this? I can't find anything explaining why...

Hi Pedro Rosmaninho,

I was not trying to use the new bead bound Tn5 for ATAC, since Illumina sells the old one especially for it. But I assume that the bead, which is relatively big, will prevent the transition of the enzyme inside the nuclei and/or inside the chromatin confinement to perform cutting events. Anyway, this is my speculation, and if you succeed in doing ATAC with this new bead-bound Tn5, would be great to hear your feedback.

Best,

Michail

Well, I haven't been able to obtain any amplification of tagmented DNA still, I have plenty of adapter dimers, but I am certain I am using the adequate number of cells at the start of the protocol (FACS sorted and then I count again with a Nieubauer chamber).

I am following the Buenrostro, and I confirmed with other published papers that the lysis buffer actually works on the cell type I am lysing.

I don't believe that the beads would affect transition to the nucleus. I expect the lysis will release all the chromatin. I will try to do the protocol without resorting to this kit probably.

Pedro Rosmaninho

Well, the best experiment will be to run side-by-side library preparation with new and old Tn5...

Actually, the nuclei is quite intact after the cell lysis. You can check it with DAPI, for example. Lysis step before the tagmentation is required to isolate the nuclei and to loosen the nuclei membrane, to let the enzyme to penetrate it and to perform cutting events. Such, if the chromatin would have been released, there would be no peaks after the sequencing, right? The Tn5 and the cut fragments even stay inside the nuclei after the tagmentation incubation. This require the additional SDS lysis step and SDS quench by Tween before PCR. Or a column cleanup. You can see this steps in the recent papers about single-cell ATAC (attached): first the bulk tagmentation is taking place, then the tagmented nuclei (with cut fragments inside) are FACS-sorted on the plate, where they are lysed by SDS and the fragments are released.

So the nuclear pore is roughly 120nm in diameter. The diameter of the bead, to which the Tn5 is attached, if we assume that this is the same bead, as SPRI (such, in Nextlex Protocol they say that they sediment only at 280g centrifugation), is in the range of hundreds (if not thousands) nanometers. Imagine, that Tn5 should also go to the small open promoters and cut it from both sides... Altogether, this is my concern why it is not working.

Best,

Michail

Article A rapid and robust method for single cell chromatin accessib...

Article Single cell RNA-seq and ATAC-seq analysis of cardiac progeni...

Yes; that actually makes a lot of sense. Also they mention in the page of the Nextera DNA Flex kit the following:

The Nextera DNA Library Prep Kit has been obsolesced. For whole-genome sequencing, Nextera DNA Flex Library Prep Kit is the recommended replacement product. 

For ATAC-seq and other custom applications, contact your local Illumina Sales representative to order stand-alone components TDE1 Tagment DNA Enzyme (Catalog No. 15027865) and TD Tagment DNA Buffer (Catalog No. 15027866).

Therefore I assume this kit is not suitable for ATAC. I'll just cut my wasted time and order the enzyme and buffer.

Just adding to this, Illumina doesn't recommend using the Flex kit with the bead bound Tn5 for ATAC. They haven't said why. If you do gather some side by side data showing that it works, it'd be great to see it posted here.

Cheers

Hi Barbara,

Basically, you even do not necessary need TD buffer - you can cook it by your own.

You need:

Tn5 transposase (from "old" nextera kit)

Lysis/Tagmentation buffer ("THS TD Buffer", see attached picture for the recipe)

Digitonin detergent (2% stock)

Qiagen PCR cleanup columns

PCR Mastermix (like NEB NebNext 2x)

Nextera i5 and i7 Indexes

SPRI Beads (or again Qiagen PCR cleanup columns)

To do Tagmentation, you spin your cells (50k) to a pellet and resuspend it in TD MasterMix, which consist of:

12,5ul 4xTHS TD Buffer

5ul 0.1% Digitonin

30ul H2O

2,5ul Tn5 Enzyme

Keep it shaking for 30mins at 37 degrees. Later do Qiagen column cleanup and proceed to PCR. Such, take 10ul of eluted tagmented DNA and mix it with 25ul 2xPCR MM, 13ul of H2O and 1ul X2 i5&i7 primers. PCR is usual (98/63/72 degrees). Usually you need 10-15 cycles, you can evaluate it by side qPCR reaction (for this you need something like SYBR-green PCR master mix). You need to perform column/bead cleanup after the PCR. I would also suggest to do the double-sided (size selection) SPRI bead cleanup, though it is optional.

Best,

Michail

Thank you, thank you Michail! I would like to do Omni ATAC-seq, because will be working with frozen liver tissue, so will follow the protocol by Corces el al. However after transposition, the reminder of the ATAC-seq library preparation was performed as described by Buenrostro et al. Do you know indexes catalog numbers?

Index kits are FC-121-1011 or FC-121-1012. However, you can also order custom indexes from your regular primer company. Feel free to contact me, if you have further questions.

PS. The protocol I mentioned also works with frozen samples, though the efficiency is lower than with fresh samples. I made frozen liver samples some years ago, worked okay.

Best,

Michail

Hi! I'm new to ATAC-seq. I wonder if any of you succeeded with the new TDE1 Tagment DNA Enzyme (Catalog No. 15027865) and TD Tagment DNA Buffer (Catalog No. 15027866) that illumina sells to replace the ones in the dicontinuated Nextera Library prep kit. I followed the Omni protocol and apparently I did not get any amplification. I would like to discard there is something wrong with those. Are they the same concentration as thay where in the kit?

Hi Yoseli Quiroga ,

The enzyme and buffer are the same as before and work fine for us. Probably there is something wrong during the library preparation. I would suggest to use the modified ATAC protocol from the Teichmann lab (see attached paper). They are using Digitonin for lysis and custom made lysis buffer (4x THS). You can check the Tagmentation part in the "Material and Methods" of attached paper.

In a nutshell ATAC is very simple:

1) Pellet cells, add lysis buffer (made with Digitonin, with Tn5 transposase), incubate 30 min at 37 deg with shaking.

2) Do column cleanup (Qiagen PCR cleanup).

3) Run PCR with Nextera primers (with optional side qPCR to evaluate the number of PCR cycles necessary).

4) Do double-sided bead cleanup.

Done!

Good luck,

Michail

Article A rapid and robust method for single cell chromatin accessib...

Hi!

Thanks for starting this discussion. I´ve earlier used the Nextera DNA lib prep kit too, so I also was´nt sure how to go about making new ATAC libraries.

Michail Yekelchyk how do proceed after using the tn5 ? Do you order oligos for indexing yourself or do you use one of the existing Illumina kits to add on indeces by PCR?

Hi Line Lieblein Røsæg ,

I am using the usual Illumina Nextera N5xx and N7xx primers. But it is just because we still have quite a lot in the stock and they last long. The custom oligos should work as well.

Best,

Michail

Just addition to what Michail Yekelchyk has said. The current Nextera primers has 10uM stock conc, which earlier used to have 25 uM (before 2018). I have tried the customized primers (made the stock of 25 uM ) and that works good. I prefer the customized over NEB ones.

Hi,

Thank you for this post! It's really helpful.

You mentioned that Illumina is now selling the Tn5 transposase (and TD buffer) separately.

Could you send me the reference or web links to these products?

Thank you very much for your help

Antonie,

TDE1 Tagment DNA Enzyme (Catalog No. 15027865) and TD Tagment DNA Buffer (Catalog No. 15027866)

They do not offer this on-line, you have to call to Illumina order them.

Thank you!

Hello all,

Some news about this ATAC-seq kits from Illumina. I also used to use the Nextera DNA-prep kit for performing ATAC-seq (up to last year). I have a new project beginning and some collaborators sent me their ATAC-seq protocol where I saw that they used the standalone enzyme (TDE1) and buffer (TD). I sent a quote request to Illumina yesterday and their answer is now that the standalone products are discontinued from September 23rd, 2019. Instead, they propose some "Illumina Tagment DNA small" and "large" kits but with a substantial price increase, compared to the Nextera kit (which also comprised some PCR primers, whereas the new kits are only TDE1 + TD). For example, I paid 2021.00 € for the Nextera kit last year and the new small kit containing the same volumes of enzymes and buffer is proposed at 3640.00 €! I really think that Illumina is abusing is monopolistic position here, any replacement suggestion?

Have a nice day!

You could use the Picelli paper and make your own Tn5.

Tn5 transposase and tagmentation procedures for massively ...

https://genome.cshlp.org › content › 2033.full.htmlby S Picelli - ‎2014 - ‎Cited by 209 - ‎Related articlesJul 30, 2014 - Tagmentation-based library construction, using the Tn5 transposase, .... more efficiently used for single-cell RNA-seq applications (Picelli et al.

To come back to my previous message and give Illumina some credit: prices have not been decided by sale people but by development teams with little, if any, field knowledge (as told by my local sales representative). As a consequence, a world-wide 50% promotional code is available (TDE50) up to March 31, 2020, the time field sales(wo)men need to settle the "right" price for the new kits. At the end I paid less for a new kit than for a Nextera DNA-prep kit ordered in 2018, so there may be some hope, even for (or from) Illumina !?

Thanks guys this discussion has been very helpful! I was a bit shocked to see the price for the TDE1 enzyme and buffer. Very happy to hear that a 50% discount is available, I hope to get an updated quote from Illumina soon.