Nuclear Isolation from Cryopreserved In Vitro Derived Blood Cells.

Induced pluripotent stem cell (iPSC)-based models are excellent platforms to understand blood development, and iPSC-derived blood cells have translational utility as clinical testing reagents and transfusable cell therapeutics. The advent and expansion of multiomics analysis, including but not limited to single nucleus RNA sequencing (snRNAseq) and Assay for Transposase-Accessible Chromatin sequencing (snATACseq), offers the potential to revolutionize our understanding of cell development. This includes developmental biology using in vitro hematopoietic models. However, it can be technically challenging to isolate intact nuclei from cultured or primary cells. Different cell types often require tailored nuclear preparations depending on cellular rigidity and content. These technical difficulties can limit data quality and act as a barrier to investigators interested in pursuing multiomics studies. Specimen cryopreservation is often necessary due to limitations with cell collection and/or processing, and frozen samples can present additional technical challenges for intact nuclear isolation. In this manuscript, we provide a detailed method to isolate high-quality nuclei from iPSC-derived cells at different stages of in vitro hematopoietic development for use in single-nucleus multiomics workflows. We have focused the method development on the isolation of nuclei from iPSC-derived adherent stromal/endothelial cells and non-adherent hematopoietic progenitor cells, as these represent very different cell types with regard to structural and cellular identity. The described troubleshooting steps limited nuclear clumping and debris, allowing the recovery of nuclei in sufficient quantity and quality for downstream analyses. Similar methods may be adapted to isolate nuclei from other cryopreserved cell types.