GSM6259910: JT, replicate 2, scRNAseq; Homo sapiens; RNA-Seq
Samples from tumor and juxta tumor were gently dissociated and enzymatically digested by an incubation of 30-40-minutes, at 37°C in agitation, in CO2-indipendent medium, supplemented by Collagenase I (2mg/mL), (Sigma) Hyaluronidase (2mg/mL) (Sigma), and DNase (25ug/mL) (Roche). Afterwards, single cell suspensions were obtained by disrupting the fragments with a syringe plunger over a 40uM cell strainer, washing with PBS (Gibco) supplemented by 2mM EDTA (Gibco) and 1% of human serum (BioWest). Cell suspension was obtained after 10' of centrifugation. After isolation of total tissue-infiltrating cells, cells were counted in CO2-indipendent medium plus 5% of FBS, washed and resuspeded in PBS. HLA-DR+CD11c+ myeloid cells from tumor and juxta tumor-infiltrating cells were FACS sorted (ARIA-BD Bioscience) and collected in  in PBS supplemented with 0.04% w/v BSA. DAPI solution (BD- Bioscience) used for dead cells exclusion. CD11c, HALDR, CD45, CD3-CD56-CD19 were included for the staining. Single-cell suspension was loaded into a Chromium Single Cell Chip (10X Genomics) according to the manufacturer's instructions. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, sorted-myeloid 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 a 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.