title: GSM6283927: PD1 -/- Sham, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: Hearts were harvested with atria removed. The tissue was minced with dissecting scissors and digested at 37°C for 45 minutes in DMEM medium containing collagenase I (450 U/mL), hyaluronidase type I-S (60 U/mL) and DNase-I (60 U/mL). The digested material was then filtered, washed and red blood cells were lysed using RBC lysis buffer. Cells suspensions were blocked with TruStain FcX (anti-mouse CD16/32) antibody and True-Stain Monocyte Blocker and incubated with Zombie Fixable Dyes (all from Biolegend) to label dead cells for 15min at RT in dark. Cells were then stained in FACS buffer with monoclonal CD45 antibodies at 4°C for 20 min. Cd45+ immune cells were isolated using cell sorter BD FACSAria II. Library was performed according to the manufacter's instructions (single cell 3' v3 protocol, 10x Genomics). Briefly, CD45+ immune 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.