Illumina NovaSeq 6000 paired end sequencing; Single-cell RNA sequencing data from murine hearts after infarction with endothelial tracing
Single-cell RNA sequencing data from murine hearts after infarction with endothelial tracing
For the lineage tracing studies, hearts were digested with Multi Tissue Dissociation Kit 2 (130-110-203, Miltenyi), as described for application of dissociation of adult mouse heart. In brief, after flushing murine hearts with HBSS, hearts were harvested, immediately minced and transferred to an enzyme mix and dissociated using the gentleMACS Dissociator as described in the manufacturer's protocol. Samples were filtered through a MACS SmartStrainer (70 μm) with 3 mL of PBS with 20% FBS. Samples centrifuged and the resultant supernatant aspirated. The cell pellet was resuspended in PEB buffer and 1× Red Blood Cell Lysis Solution to remove erythrocytes. Following erythrocyte removal, Enzyme A of the Multi Tissue Dissociation Kit 2 was added to PBS in a fresh tube and then combined with the cell pellet. Following this step, cells were resuspended in Dead Cell Removal MicroBeads (130-090-101, Miltenyi). Samples were mixed and incubated following Miltenyi's protocol instructions. We made the positive selection with a column of type MS (for up to 107 dead cells and up to 2×108 total cells). Prior to its use, the column was rinsed with binding buffer. Effluent was collected as the live cell fraction. Cells were counted using Trypan blue and a Neubauer chamber. Cells were then resuspended appropriately for loading on the 10X Genomics Chromium Controller chip.  Resulting non-cardiomyocyte cellular suspensions were loaded on a 10X Chromium Controller (10X Genomics) according to manufacturer's protocol. Murine scRNA-seq libraries were prepared using Chromium Single Cell 3′ v3 Reagent Kit (10X Genomics), according to manufacturer's protocols. Briefly, the initial step consisted in generating cell partitioning droplets where individual cells were isolated together with gel beads coated with unique primers bearing 10X cell barcodes, UMI (unique molecular identifiers) and poly(dT) sequences. Reverse transcription reactions were engaged to generate barcoded full-length cDNA followed by the disruption of emulsions using the recovery agent and cDNA clean up with DynaBeads MyOne Silane Beads (37002D, Thermo Fisher Scientific). Bulk cDNA was amplified using a Biometra Thermocycler Professional Basic Gradient with 96-Well Sample Block (98 °C for 3 minutes; cycled 14×: 98 °C for 15 s, 67 °C for 20 s, and 72 °C for 1 minute; 72 °C for 1 minute; held at 4 °C). Amplified cDNA product was cleaned with the SPRIselect Reagent Kit (B23318, Beckman Coulter). Indexed sequencing libraries were constructed using the reagents from the Chromium Single Cell 3′ v3 Reagent Kit as follows: fragmentation, end repair and A-tailing; size selection with SPRIselect; adaptor ligation; post-ligation cleanup with SPRIselect; sample index PCR and cleanup with SPRI select beads. Library quantification and quality assessment was performed using Bioanalyzer Agilent 2100 using a High Sensitivity DNA chip (5067-4627 Agilent Genomics).