title: GSM7310381: anti-PD-1, replicate 1, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: Tumor samples were collected and placed in pre-cooled PBS to remove necrotic tissue, blood clots, and surrounding normal tissue. The samples were then cut into small pieces and added to a 6-well plate containing 2 mL of preheated tissue lysate (5 mg/mL collagenase IV and 1 mg/mL DNase, incubated at 37 °C on a shaker at 90-100rpm for 1 hour. Next, the cell suspension was filtered using a 70µm filter, and centrifuged at 300g, 4°C for 5 minutes. The supernatant was discarded, and the cell pellet was enriched by mouse CD45 MicroBeads (Miltenyi, Cat# 130-052-301) according to the manufacturer's instructions. Enriched cells were then stained with 7-AAD and APC-Cy7 anti-mouse CD45 antibody. Alive CD45+ cells were sorted using a BD FACSAria™ III cell sorter.  Sorted alive CD45+ cells were washed in HBSS-/- and resuspended in HBSS-/- supplemented with 0.04% w/v BSA. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, tumor-infiltrating CD45+ 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.