title: GSM4085515: melanoma_p2_frozen; Homo sapiens; RNA-Seq
library_construction_protocol: Fresh tumor tissue was dissociated by manual mincing followed by an incubation of 30 min at 37°C in RPMI with liberase (0.83mg/ml; Sigma-Aldrich), alternated with 3 rounds of dissociation with a gentleMACSTM dissociator (Miltenyi). After dissociation, cell suspensions were filtered with a 100 μm filter, and resuspended in ACK lysis buffer before washing with RPMI-5% FBS. Cells were then washed with PBS and stained with LIVE/DEAD Fixable Ghost Dye™ Red 780 followed by anti-CD45.  Viable immune cells were sorted on a FACS Aria II sorter (BD Biosciences). Human CD45+ cells from human melanoma samples were FACS-isolated and short term stored at -80C in Bambanker prior to single cell RNA-seq. The scRNA-seq libraries were prepared following the user guide manual (CG00052 Rev E) provided by the 10X Genomics and Chromium™ Single Cell 3' Reagent Kit (v2). Briefly, samples were encapsulated in microfluidic droplets at a dilution of ~70 cells/µl (doublet rate ~3.9%.). Encapsulated cells were subjected to reverse transcription (RT) reaction at 53ºC for 60 min. After RT step, the emulsion droplets were broken and barcoded-cDNA was purified with DynaBeads, followed by 14-cycles of PCR-amplification (98 °C for 180 s; [98 °C for 15 s, 67 °C for 20 s, 72 °C for 60 s] x 12-cycles; 72 °C for 60 s). 50 ng of PCR-amplified barcoded-cDNA was fragmented with the reagents provided in the kit and purified with SPRI beads to obtain an average fragment size of 600 bp. Next, the DNA library was ligated to the sequencing adapter followed by indexing PCR (98 °C for 45 s; [98 °C for 20 s, 54 °C for 30 s, 72 °C for 20 s] x 10 cycles; 72 °C for 60 s). The resulting DNA library was double-size purified (0.6-0.8X) with SPRI beads and sequenced on an Illumina NovaSeq platform  (R1 – 26 cycles, i7 – 8 cycles, R2 – 96 cycles) resulting in 184.5-186.1 million reads per sample (average reads per single-cell being 42,000 and average reads per transcript 4.40-7.14).