GSM5014496: Endogenous and in vitro cardiomyocyte maturation reference; Mus musculus; RNA-Seq
For isolation of endogenous CMs from e14-p4 timepoints, we used the neonatal cardiomyocyte isolation kit from Miltenyi Biotec in conjunction with the gentleMACS Dissociator. For later timepoints, Langendorff perfusion was performed based on established literature protocols using collagenase and protease. We used a horizontal (i.e. non-hanging) Langendorff apparatus with a chamber filled with perfusion buffer. To perform isolation, we first performed isofluorane anaesthesia on non-heparinized mice. Mice were observed until clearly anaesthetized and unresponsive to toe pinch, and subsequently euthanized by cervical dislocation. The heart was then rapidly excised from the chest and cannulated to the Langendorff apparatus. We flowed digestion buffer for 9 minutes at 1.5 mL/min. Subsequently, the ventricle was excised and minced. We filtered isolated cells through a 100 µM screen to eliminate large tissue chunks, spun down at 800 RPM for 1 minute (Eppendorf centrifuge 5702), and resuspended cells in 10 mL Tyrode's buffer. All cells were isolated using LP-FACS, as outlined in Kannan et al. (Circ Res 2019).    PSC-CMs were dissociated from culture flasks with TrypLE (5-10 minutes, depending on the timepoint). Cells were strained to remove clumps and clusters and subsequently sorted into prepared collection plates on either a MoFlo Legacy or MoFlo XDP. We performed sequencing using the mcSCRB-seq protocol. The protocol has been described at protocols.io at dx.doi.org/10.17504/protocols.io.p9kdr4w. We sequenced the final, pooled library on a NovaSeqS4 as 150-base pair paired-end reads. We subsequently demultiplexed into two files such that the read 1 file contains the 8 base pair i7 tag, 6 base pair cell barcode, and 8 base pair UMI, while read 2 contains the 150 base pair cDNA read. We have provided these final demultiplexed reads to GEO. However, if the original paired-end reads from the sequencer are desired, we are happy to provide on request.