The assay for transposase-accessible chromatin with sequencing (ATAC-Seq) is a popular method for determining chromatin accessibility across the genome. By sequencing regions of open chromatin, ATAC-Seq can uncover how chromatin packaging and other factors affect gene expression, providing valuable insights into genome regulation.
The isolation of nuclei is crucial to generate quality data in ATAC-Seq. A good nuclei suspension should be clump and debri free. Nuclei sorting is often used as non-sorted broken nuclei can lead to a higher background after sequencing. Nuclei sorting also reduces contamination by organellar genomes (1). Before committing to sequencing, quality of nuclei is often assessed via visual inspection assessing shape and trypan blue staining. An intact nucleus generally appears round and smooth, in contrast to compromised nuclei that can appear ruffled and blebbing.
Whilst a standard fluorescent enabled cell sorter can isolate nuclei from cellular debris, the selection of intact nuclei cannot be performed because of the lack of imaging capabilities. Here we propose that high-speed fluorescence image-enabled sorting previously described by Schraivogel et al (2) can enable sorting of higher quality nuclei. The eccentricity parameter provides a measurement of roundness of the nuclei that can be used to determine the sort decision outcome. In our study with nuclei isolated from mouse liver cells, we were able to identify elongated/less-round looking nuclei with eccentricity. We have also sorted & compared round vs. less-round nuclei. In our preliminary results, the sort recovery rate is lower with less-round nuclei. DNA extracted from less-round nuclei also shows higher fragmentation.