Dear All,

I have recently started to work on ATAC-seq and for this purpose I need nuclei as pure as can be prior to transposition and so on.

After homogenization by douncing. I am actually using an Optiprep gradient for that and centrifuging at 10000g for 25 min. Unfortunately, in what is supposed to be the nuclear fraction, I also find some cell clusters and other debris.

Prior to gradient centrifugation, I have a pre-centrifuge at 100g for 1 min to pellet the debris. I tried to increase that speed to 300, but in that case I could not see a lot of nuclei in the final fraction after UC.

I was wondering if using a 40µm strainer can help to give a rough separation in between clusters anc pure nuclei.

Clues are always accepted!

N.B the lysis is performed during douncing with a buffer containing NP-40

Thank you!