GSM8268885: Young PD-1, scRNA-seq; Mus musculus; RNA-Seq
Single cells were harvested from the lung of tumor-beairng young or aged mice by dissociating with collagenase D and Dnase I, and then mononuclear cells were enriched by density gradient centrifugation. CD45+ immune cells were labeled with biotin-conjugated anti-CD45 Ab and isolated by EasySep Release Mouse Biotin Positive Selection Kit (STEMCELL TECHNOLOGIES). Magnetic particles bound to CD45+ cells were removed after the procedure. Viable cells were further separated by passing throuhg 40 microm cell strainer and using  Dead cell remover (Miltenyi Biotech) according to the manufacturer's insturction. Library construction was performed according to the manufactures' instructions (single cell 3' v3 protocol, 10x Genomics). Briefly, CD45+ cells in the lung were resuspended in the master mix and loaded together with patitoning 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 Truseq Read1 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 apropriated sized fragments were selected with SPRIselect reagent for subesquent library construction. During the library construction TruSeq Read2 sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.