title: GSM8060144: Left ventricle, male, sham TAC surgery, 14dpi; Mus musculus; RNA-Seq
library_construction_protocol: Following excision of the mouse heart, the atria and atrioventricular valves were surgically removed. We then dissected the left and right ventricle free walls, and washed the tissue in chilled DPBS. Remaining septal tissue was discarded. LV and RV tissue samples were then minced and digested in a solution of 2 mg/ml Collagenase type II (Worthington Cat#: LS004176) and 2 mg/ml Dispase II (Sigma Cat#: D4693) at 37°C for 45 minutes. Trituration was performed with a 10mL serological pipette at the 15, 30, and with a 1 mL pipette at the 45 minute mark. Samples were then filtered through 70 µm strainers into 30mL of chilled DPBS, split into two 15mL Eppendorf tubes, and centrifuged for 7 min at 500g. . After decanting, pellets were resuspended in 100uL of Hank's Balanced Salt Solution (HBSS containing 2% FBS, and incubated with CD31 and CD45 magnetic beads for 15 minutes. Endothelial (CD31+) and hematopoietic cells (CD45+) were subsequently removed using the AutoMacs instrument (Miltenyi Biotec). The remaining cells were stained with DAPI, and live CFs (GFP+, DAPINeg) were sorted on the Aria IIu (BD Biosciences) into HBSS containing 2% FBS for preparation for scRNA-seq. FACS-isolated CFs were manually counted using a hemocytometer before single-cell library preparation using the 10x Genomics Chromium system. Library preparation and sequencing were performed at The Centre for Applied Genomics at the Peter Gilgan Centre for Research and Learning. Approximately 10,000 cells were loaded on the 10X Genomics Chromium Controller instrument to generate single-cell GEMs (gel beads in emulsion) using the Chromium Single Cell 3ʹ Reagent Kits v3 kit (part -1000092, 10X Genomics, Pleasanton, CA) following the manufacturer's instructions.  Briefly, barcoded full-length cDNA is first produced from poly-adenylated mRNA.  cDNA is then fragmented and made into Illumina-ready libraries.  The final libraries contain a 10X barcode, UMI, sample index and Illumina P5 and P7 sequences.  Size, quality and concentration of the cDNA and corresponding 10X library were evaluated by the Agilent 2100 Bioanalyzer system and quantified using the Kapa Library Quantification Illumina/ABI Prism Kit protocol (KAPA Biosystems).   Libraries were sequenced on the Illumina NovaSeq6000 instrument on the S4 flowcell using 29+8+101 sequencing configuration to generate 500M reads of data per sample.