Hi,

Since couple of months, I am working on ATAC-seq optimization starting from mouse brain tissues using Buenrostro et al Protocol (Curr. Protoc. Mol. Biol., 2015). At the end of library preparation, and just prior to sequencing, I am trying to validate the established library using qPCR by checking the enrichment of accessible and inaccessible regions known to be found in the mouse brain tissues (For this aim I designed a set of positive and negative primers based on publicly available ATAC-seq data from the ENCODE project). The issue I am facing is that both negative and positive primers are giving exactly the same enrichment level. I checked every single step in the protocol and so far it is going in line with Buenrostro's protocol. Actually, I am doubting the integrity of nuclei being isolated from the brain tissues (noting that for nuclei isolation I am using a commercial kit from invent biotechnologies). I am checking the quality of nuclei using Trypan blue counting method and morphologically wise, they look good. But actually, I am not sure about the chromosomal integrity of those nuclei and whether the former is affected by the procedure of isolation.

I would greatly appreciate if someone can share his/her experience in this issue or suggest something in this concern.

Thank you in advance,