RNA-seq of homo sapiens: adult cardiomyocyte nuclei
Single nuclei were isolated from snap-frozen mouse and human left ventricle and processed by mechanical dissociation at 4000?Hz (4??20?s pulses) in Lysonator cartridges (SG Microlab devices) and counterstained with DAPI. CM nuclei were stained with PCM1 antibody (1:500, HPA023374, Sigma), secondary anti-rabbit Alexa 488 (1:500) or Alexa 568 antibody (1:500), and captured individually using C1 Single Cell Auto Prep system (1017?uM mRNA seq chip, Fluidigm). Automated imaging of captured nuclei was performed on an inverted microscope (Olympus) with 10 objective (Olympus) and CCD camera (Axiocam MR3, Zeiss) to confirm the identity of wells containing only single PCM1+ CM captured. PCM1+ CM nuclear RNA-seq libraries were prepared using Nextera XT DNA sample preparation kit (Illumina). Each sample was sequenced with paired end 2??101?bp reads on HiSeq 2500 (Illumina).