GSM6008295: CD31Neg Non-myo, replicate 1, scRNAseq; Mus musculus; RNA-Seq
GSM6008295
The heart was removed by cutting the heart below the atrium and residual atrial tissue was trimmed off after removal. In order to remove residual blood, ventricular tissue was washed in HBSS  (without calcium or magnesium) several times, then cut into large pieces in ice cold HBSS, and placed into ice cold HBSS for up to 15 minutes. Once harvest of ventricular tissue was completed, the tissue was removed from HBSS, wet with a digestion buffer, and minced into fine particles with a scalpel. Digestion buffer consisted of collagenase IV and dispase II at a concentration of 2 mg/mL and 1.2 U/mL. Tissue was collected and each heart was placed into a 3 mL digestion buffer, where it was then incubated in 15 mL conical tubes in a 37 degree water bath. Following 15 minutes of digestion, the digestate was triturated with a 1000 uL pipette tip, and then digested for another 15 minutes. The digestion was kept at 30 to 45 minutes, depending on particle size and completeness of digestion. After digestion, cells were strained, and the strainer was washed with ice cold HBSS with 2% FBS, until a final volume of 10 mL was obtained. This suspension was washed one more time with ice cold HBSS, and then incubated in 5 mL of ACK lysis buffer for 5 minutes at room temperature to induce lysis in remaining erythrocytes. The reaction was quenched by adding an equal volume of ice cold HBSS with 2% FBS, washed once more with HBSS with 2% FBS and then a MACS depletion or debris removal step was performed. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, GCs 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.