GSM4628096: YAP5SA Drop-Seq 2; Mus musculus; RNA-Seq
Adult hearts from indicated genotypes were dissected, cannulated, and then dissociated into a single-cell suspension via collagenase digestion (Collagenase A, Roche) on a custom built Langendorff apparatus as described previously (Monroe et al., 2019) with minor modifications. In short, we performed cardiomyocyte enrichment whereby cardiomyocytes were allowed to settle by gravity for 5 minutes at room temperature after washes in a calcium free Tyrode's buffer and the supernatant, containing non-CMs was put into new tube and spun down at 700 x g and the pellet containing CMs was washed and spun down at 300 x RPM. Finally, cells were combined in the final step and counted prior to dilution and input into Drop-seq rig. We also performed CM-enrichment batches whereby only the CM enriched fractions were used for Drop-seq. Dissociated cells were diluted to a concentration of 200 cells per µL in PBS with 0.01% BSA. Drop-seq was then performed according to (Macosko et al., 2015). Here cells were co-encapsulated into nano-liter sized droplets containing barcoded microparticles (ChemGenes, catalog number Macosko201110) and lysis buffer using a custom microfluidics device (FlowJEM, Toronto, Canada). After droplet breakage, reverse transcription (Thermo), and exonuclease treatment (NEB) all cDNA was PCR amplified (KAPA), pooled, purified with Ampure XP beads (Beckman Coulter), and ran on a Fragment analyzer (Advanced Analytical Technologies, Inc.) for quality control, quantification, and size determination. Library preparation was performed with the Illumina Nextera XT kit, and libraries were triple purified with Ampure XP beads (Beckman Coulter). All libraries were sequenced on an Illumina NextSeq500 instrument.
GEO Accession
GSM4628096