title: GSM6634871: scB2 (P3),scRNAseq; Homo sapiens; RNA-Seq
library_name: GSM6634871
library_construction_protocol: Resected OSCC-GB tissue samples were collected in pre-chilled MACS tissue storage solution (Milteny Biotec) and transported on ice immediately after surgical procurement. The Freshly collected tissue was then cut into small pieces ≤ 1-2 mm with sterile scissors in a dish containing 1 mL of enzyme mix (enzymes H, R, and A from Miltenyi Biotech in RPMI1640 medium). The minced tissue suspension and the remaining enzyme mix were transferred to a gentleMACS C tube and then placed onto the gentleMACS Octo dissociator (Miltenyi Biotech). The GentleMACS program 37C_h_TDK_3 was run for dissociation after attaching the heating elements. Following completion of the program, the homogenate was briefly centrifuged and filtered through a 70-μm cell strainer (BD Bioscience) into a 50-mL centrifuge tube (Axygen). The strainer was then rinsed with 15 mL of RPMI1640 medium (Gibco) and the cell suspension was then centrifuged at 300g for 7 min. The supernatant was aspirated and the pellet was resuspended in RPMI1640 medium.  Red blood cells in the cell pellet were lysed by incubating the pellet in RBC lysis buffer (Miltenyi Biotec) for 10 minutes at 4°C. Then, 10 ml of chilled RPMI1640 medium was added and the tube was centrifuged at 300g for 10 min at 4°C. The supernatant was then aspirated and the cell pellet was resuspended in RPMI1640 medium.  The number of cells and fractions of live cells in suspension was counted using trypan blue and Live/Dead Viability/Cytotoxicity Kit (Thermo Fisher Scientific) in Countess II FL Automated Cell Counter (Thermo Fisher Scientific). The scRNA-seq library was generated using single cell 3' v3 protocol, 10x Genomics according to the manufacturer's instructions. Briefly, isolated single 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 appropriated-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.