Dataset Information

ATAC-seq on mouse embryonic stem cells for asynchronous cells, mitotic cells and purified mitotic chromosomes.

ABSTRACT: E14Tg2a mouse embryonic stem cells (ESCs) were cultured on gelatin coated plates in KO-DMEM medium supplemented with 15% FCS and 1000 U/ml leukaemia inhibitory factor. Rapidly dividing cultures of ESCs were arrested in metaphase using demecolcine (0.1 μg/ml for 6 h), achieving samples where most (85-90%) cells were in M-phase as judged by propidium iodide labelling. Condensed chromosomes were released using polyamine buffer, stained with Hoechst 33258 and Chromomycin A3 and purified by flow cytometry using a Becton Dickinson Influx equipped with specialised air-cooled lasers.

ORGANISM(S): Mus musculus

PROVIDER: GSE147552 | GEO | 2020/06/14

REPOSITORIES: GEO

ACCESS DATA

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Project description:Normal donors BM CD34+ cells were differentiated in thrombopoietin-treated liquid suspension cultures. Briefly, CD34+ cells (80,000 /mL) were resuspended in a serum-free medium in the presence of 100 ng/mL of human recombinant thrombopoietin (TPO; Genzyme, Boston, MA). Every 3 days, viable cells were scored by Trypan blue dye exclusion and cultures were amplified with fresh serum-free medium. Each well was then supplemented with 100 ng/mL TPO. After 14 to 16 days of liquid culture, CD34-derived megakaryocytes (MKs) were purified by means of an anti-CD41a monoclonal antibody (MoAb) (Dako, Milan, Italy) directed against the glycoproteic *IIb-*3 complex and immunobeads (MPC 450 Dynabeads; Dynal, Oslo, Norway), as previously described (27). The purity of MKs was determined for each isolation by indirect immunofluorescence, using an anti-CD41b MoAb which reacts with a different epitope of *IIb-*3 subunit (Immunotech Inc., Westbrook, ME), followed by a goat anti-mouse IgG, covalently linked to fluorescein (Becton Dickinson, San Jose, CA). Essential Throbocytemia BM CD34+ cells were differentiated in thrombopoietin-treated liquid suspension cultures. Briefly, CD34+ cells (80,000 /mL) were resuspended in a serum-free medium in the presence of 100 ng/mL of human recombinant thrombopoietin (TPO; Genzyme, Boston, MA). Every 3 days, viable cells were scored by Trypan blue dye exclusion and cultures were amplified with fresh serum-free medium. Each well was then supplemented with 100 ng/mL TPO. After 14 to 16 days of liquid culture, CD34-derived megakaryocytes (MKs) were purified by means of an anti-CD41a monoclonal antibody (MoAb) (Dako, Milan, Italy) directed against the glycoproteic *IIb-*3 complex and immunobeads (MPC 450 Dynabeads; Dynal, Oslo, Norway), as previously described (27). The purity of MKs was determined for each isolation by indirect immunofluorescence, using an anti-CD41b MoAb which reacts with a different epitope of *IIb-*3 subunit (Immunotech Inc., Westbrook, ME), followed by a goat anti-mouse IgG, covalently linked to fluorescein (Becton Dickinson, San Jose, CA). Keywords: other

Dataset Information

ATAC-seq on mouse embryonic stem cells for asynchronous cells, mitotic cells and purified mitotic chromosomes.

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets