Homo sapiens
Nuclei from the left ventricle of Donor2, CBTM-362C
Biomaterial type
cell_suspension
SRA accession
ERS5531312
Sample Name
CBTM-362C_LV_nuclei_2
cell_morphology - cell_viability_method
Trypan Blue
dissociation_protocol - method
enzymatic dissociation
dissociation_protocol - protocol_core - protocol_description
Single nuclei are obtained from flash-frozen tissues using mechanical homogenization as previously described. Tissues are homogenized using a 7 ml glass Dounce tissue grinder set (Merck) with 8-10 strokes of a loose pestle (A) and 8-10 strokes of a tight pestle (B) in homogenization buffer (250 mM Sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Tris-HCl, 1 mM dithiothreitol (DTT), 1x Protease Inhibitor, 0.4 U/µl RNaseIn, 0.2 U/µl SUPERaseIn, 0.1% Triton X-100 in nuclease-free water). Homogenate is filtered through a 40 µm cell strainer (Corning).
dissociation_protocol - protocol_core - protocol_id
sn_dissociation
dissociation_protocol - protocol_core - protocol_name
Single Nuclei dissociation of human heart tissue protocol
dissociation_protocol - protocol_core - protocols_io_doi
10.17504/protocols.io.veae3ae
dissociation_protocol - protocol_core - publication_doi
10.1101/2020.04.03.024075
enrichment_protocol - markers
DAPI+
enrichment_protocol - method
fluorescence-activated cell sorting
enrichment_protocol - protocol_core - protocol_description
After centrifugation (500×g, 5 min, 4 °C) the supernatant is removed and the pellet is resuspended in storage buffer (1x phosphate-buffered saline (PBS), 4% bovine serum albumin (BSA), 0.2 U/µl Protector RNaseIn). Nuclei are stained with NucBlue Live ReadyProbes Reagents (ThermoFisher) and Hoechst-positive single nuclei are purified by fluorescent activated cell sorting using Influx, XDP or FACSAria (BD Biosciences). Nuclei purification and integrity is verified under microscope
enrichment_protocol - protocol_core - protocol_id
nuclei_purification
enrichment_protocol - protocol_core - protocol_name
Nuclei purification
enrichment_protocol - protocol_core - protocols_io_doi
10.17504/protocols.io.veae3ae
estimated_cell_count
5000