GSM7290658: CCS, scRNAseq; Mus musculus; RNA-Seq
P1, P2, and P4 neonatal cardiomyocytes were isolated by enzymatic digestion with 0.075% Collagenase II/Dispase. Isolated cardiomyocytes were suspended in culture medium (DMEM/F-12 with 10% FBS + 1% P/S) were filtered using a 100 uL cell strainer (VWR, 10199-658), then transferred to separate polypropylene tubes (Falcon). Cells were incubated with SytoxTM Blue dead cell stain (Thermofisher, S34857, 1:2000) prior to Fluorescence Activated Cell Sorting (FACS). Gating was done for size and granularity, then for Sytox-based viability, and finally for tdTomato-based identification of conduction cells. Approximately 0.1-0.2% of total viable cardiomyocytes were tdTomato-positive cells for all P1, P2, and P4 cells.   MULTI-seq reagents were generously provided by the developers, and multiplexing of P1, P2, and P4 cells was conducted following the developer's protocols. In brief, following separate FACS sorting of P1, P2, and P4 CCS cells into culture medium (DMEM/F-12 with 10% FBS + 1% P/S ), cells were pelleted and washed using centrifugation (250 x g, 5 min, 4C) and 1x PBS.  Lipid-modified oligonucleotide anchor mix and unique barcodes were added to each sample at final concentrations of 200 nM. Cells were resuspended by gentle pipetting and were incubated at 4C for 10 min, ensuring to periodically mix by gently tapping. Then, a co-anchor lipid-modified oligonucleotide was added to each sample (final concentration of 200 nM), and cells were incubated at 4C for 5 min. Cells were then pelleted using centrifugation (250 x g, 5 min, 4C) and resuspended in PBS containing 1% BSA to quench residual barcodes. P1, P2, and P4 cells were then pooled into a single tube and subjected to viability assessments and cell counting prior to 10x Genomics scRNA-seq library generation using the Single Cell 3' v3 kit. Before library generation, P1, P2, P4 cells were barcoded using MULTI-seq reagents. Then P1, P2, and P4 cells were pooled and library generation was performed according to the manufacter's instructions (single cell 3' v3 protocol, 10x Genomics). Briefly, cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added. scRNA-seq + MULTI-Seq