CUT & RUN to Profile Chromatin-Bound Proteins in Primary Mouse Neural Progenitor Cells.

Cleavage under targets and release using nuclease (CUT & RUN) is an innovative method to profile histone modifications and chromatin-bound proteins genome-wide. CUT & RUN offers two distinct advantages of requiring much fewer cells and providing strong signal-to-noise ratios in deep-sequencing data. Here, we describe a workflow starting from dissociation and sorting of mouse embryonic brains, CUT & RUN, and DNA library preparation to deep sequencing. With our workflow, researchers can obtain high-quality sequencing data to profile histones and chromatin-associated proteins by using as few as 100,000 neural progenitor cells (NPCs).

A complete workflow for the differentiation and the dissociation of hiPSC-derived cardiospheres.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) are an invaluable tool for both basic and translational cardiovascular research. The potential that these cells hold for therapy, disease modeling and drug discovery is hampered by several bottlenecks that currently limit both the yield and the efficiency of cardiac induction. Here, we present a complete workflow for the production of ready-to-use hiPSC-CMs in a dynamic suspension bioreactor. This includes the efficient and highly reproducible differentiation of hiPSCs into cardiospheres, which display enhanced physiological maturation compared to static 3D induction in hanging drops, and a novel papain-based dissociation method that offers higher yield and viability than the broadly used dissociation reagents TrypLE and Accutase. Molecular and functional analyses of the cardiomyocytes reseeded after dissociation confirmed both the identity and the functionality of the cells, which can be used in downstream applications, either as monolayers or spheroids.

Quick and reliable method for retina dissociation and separation of rod photoreceptor perikarya from adult mice.

A pure and abundant population of adult rod perikarya can be exploited in different studies concerning nuclear functions such as gene expression analyses which aim at elucidating the relationship between cell type and disease [1]. Sorting is based either on specific cell-surface markers or fluorescently labeled reporter proteins. Here, we describe a simple and reliable method for separation of rod photoreceptor perikarya without the use of staining procedures or transgenic mice. This method is limited, however, to sorting rod photoreceptors from adult mouse retina. Mature rods possess an inverted nuclear architecture which is determined by the optical functions of these nuclei [2]. The high backscatter of heterochromatin in the core of the nucleus can be used as a selection criterion for FAC-sorting by forward and sideward scatter. •The procedure for retina dissociation using the Papain Dissociation System (Wothington Biochemical Corporation) was optimized.•An easy to follow step-by-step protocol for retina dissociation was devised.•Rod perikarya were FAC-sorted by forward and sideward scatter based solely on the high backscatter of heterochromatin in their nuclei.