[pabanga1]

FWIW I got this working. I adopted the OMNI-ATAC protocol and get great library amplification from 5k cells using only 2% the amount of Tn5 required for 50k cells. Adding a protease inhibitor during transposition was crucial for my library prep.

Hi! I am wondering what does exactly from the Omni-ATAC protocol mean:

"Add 50 ul cold ATAC-Resuspension Buffer (RSB) containing 0.1% NP40, 0.1% Tween-20, and 0.01% Digitonin and pipette up and down 3 times.
Incubate on ice for 3 minutes.
Wash out lysis with 1 ml of cold ATAC-RSB containing 0.1% Tween-20 but NO NP40 or digitonin and invert tube 3 times to mix"

Does this mean I need to removed the previous 50 uL and wash with cold ATAC-RSB?

[Rosmano]

Hi! I am wondering what does exactly from the Omni-ATAC protocol mean:

"Add 50 ul cold ATAC-Resuspension Buffer (RSB) containing 0.1% NP40, 0.1% Tween-20, and 0.01% Digitonin and pipette up and down 3 times.
Incubate on ice for 3 minutes.
Wash out lysis with 1 ml of cold ATAC-RSB containing 0.1% Tween-20 but NO NP40 or digitonin and invert tube 3 times to mix"

Does this mean I need to removed the previous 50 uL and wash with cold ATAC-RSB?

This means you add 1mL of cold ATAC-RSB containing 0.1% Tween-20 to the 50uL to wash the sample

[Rosmano]

I always do both upper and lower size selection using AMPure XP beads, I don't see a reason not to just do it right away (unless you are unfamiliar with bead cleanup and want to have a way to check you process).
I add 0.5x, keep supernatant, add 1.3x (total 1.8), keep beads, elute. Then check BA.

I attached some BA tracks from a test I did, D and E are how I like my libraries to look and these two sequenced nicely.

I did a left side size selection and got the following band in the Fragment Analyzer. I stored my samples at -20ºC, so now I am going to do the right-side size selection as you describe. I was hoping that just doing the left-side size selection would be enough, because I did a mock experiment using unsorted cells to see how it would work.

Do you think that there's an issue in thawing my samples now and do the right size selection?

[Meyana]

I did a left side size selection and got the following band in the Fragment Analyzer. I stored my samples at -20ºC, so now I am going to do the right-side size selection as you describe. I was hoping that just doing the left-side size selection would be enough, because I did a mock experiment using unsorted cells to see how it would work.

Do you think that there's an issue in thawing my samples now and do the right size selection?

I can't see why there should be any problem in doing the upper size selection now, I would just try and see how it sequences.

Hope it works.

[Rosmano]

Oh, just an update: it worked beautifully!

I prepared a few new libraries. Could you guys provide feedback and tell me what do you think of these new samples?

https://ibb.co/nbWRn1p

https://ibb.co/RNF2vd4

https://ibb.co/xjKBPth

[Pab77]

Hi Rosmano, These look amazing! I think all of these should give you good resolution. Did you use 50K cells, protease inhibitor, and double-sided size selection? I've been working on C. elegans ATAC-seq with a titration of nuclei but not getting the nice laddering. For C. elegans, several papers used ~1-2 million nuclei for ATAC-seq library prep. However, not working in our hands. I'm using Buenstrostro protocol. Any input would be greatly appreciated.

[Charbelfeg]

Hello Rosmano,

I am having the same problem as you. I obtain no CT at the qPCR stage. I am using NEB master MIX. Can you please share what did you do?

[ASadiasa]

Hi! I am fairly new with ATAC sequencing and had the following libraries. Can you please tell me if there ar any problems here?
The sequencing facily have advised us that the libraries look good and as expected so we pushed through with the sequencing. However, we found a very low amount of peaks after analysis. One reason given to us is that the samples may have been overtagmented. Thank you very much!

[Rosmano]

You seem to have some very large fragments in there. Did you do right side selection?