Sample 2
Mus musculus
Protocols: Mice were fed standard diet ad libitum prior to isolation of tissues. Male and female mice were sacrified at 10 weeks of age. Single cell suspensions from isolated mouse hearts were prepared by tissue mincing and enzyme digestion. Briefly, mice were euthanized by CO2 asphyxiation, before perfusion with Perfusion Buffer (1 × DPBS, 0.8 mM CaCl2) until complete blanching of the liver (~7 minutes). Subsequently, hearts were isolated, atria and valves removed, and ventricles minced to ~1 mm cubes. Minced tissue was digested in 3 mL of Digestion Buffer (2 mg/mL Collagenase IV [Worthington Biochemical], 1.2 units/mL Dispase II [Sigma-Aldrich], in Perfusion Buffer) for 45 minutes at 37° C with tissue suspension triturated every 15 minutes using 1000 µl micropipettes. The resulting cell suspension was filtered through a 70 µm filter to remove residual undigested tissue pieces, and diluted in 15 mL Perfusion Buffer before pelleting cells at 200 × G for 20 minutes with centrifuge brakes disengaged. Cell supernatant was aspirated and the pellet re-suspended in 15 mL of 1 × HBSS (Sigma-Aldrich) + 0.8 mM CaCl2 before pelleting cells again as described above. The final cell pellets were resuspended in 250 uL of 2% FBS/HBSS. For each sample, ~12,000 cells were loaded for capture onto the Chromium System using the v2 single cell reagent kit (10X Genomics). Following capture and lysis, cDNA was synthesized and amplified (12 cycles) as per manufacturer's protocol (10X Genomics). Amplified cDNA was used to construct a sequencing library according to the manufacturer's instructions (Illumina).
ENA-FIRST-PUBLIC
2019-05-02T17:03:59Z
ENA-LAST-UPDATE
2019-05-01T08:20:57Z
External Id
SAMEA5587293
INSDC center name
Jackson Laboratory
INSDC first public
2019-05-02T17:03:59Z
INSDC last update
2019-05-01T08:20:57Z
INSDC status
public
Submitter Id
E-MTAB-6173:Sample 2
age
10
broker name
ArrayExpress
cell type
cardiac non-myocyte
common name
house mouse
developmental stage
adult
genotype
wild type genotype
organism part
cardiac ventricle
sample name
E-MTAB-6173:Sample 2
scientific_name
Mus musculus
sex
mixed
strain
C57BL/6J