GSM5163837: NCM SS2 sham1_24; Mus musculus; RNA-Seq
For the isolation and collection of non-cardiomyocytes, hearts were minced and enzymatically dissociated using 2 mg/mL type 2 collagenase (Worthington), 1 mg/mL dispase (Roche), and 20 U/mL DNAse I (Roche), with 5 cycles of digestion for a total 40 min at 37 °C. After using a 40-μm cell strainer (Greiner) to remove the cardiomyocytes, cells were stained with Zombie Green Fixable Viability Kit (BioLegend) and live cells were collected by fluorescence-activated cell sorting (FACS) using a FACSJazz cell sorter (BD Biosciences). Single-cell cDNA libraries were generated using the Smart-seq2 protocol. For the cDNA libraries constructed by Smart-seq2, the efﬁciency of reverse transcription was assessed by examining the cycle threshold (Ct) values of control genes (Tnnt2 for cardiomyocytes) from quantitative real-time polymerase chain reaction (qRT-PCR) using a CFX96 Real-Time PCR Detection System (Bio-Rad), and the distribution of cDNA fragment lengths was assessed using LabChip GX (Perkin Elmer) and/or TapeStation 2200 (Agilent Technologies). The following primer sets were used for qRT-PCR: Actb mRNA forward: CAACTGGGACGACATGGAGA; and Actb mRNA reverse: GCATACAGGGACAGCACAGC. A Ct value of 25 was set as the threshold.