GSM5768274: ovCHA034; Homo sapiens; RNA-Seq
The resected tissues were cut into blocks approximately 1cm in diameter. The premixed tumor dissociation working solution (Miltenyi Biotec, Germany) was added to minced tissue. The tissue and working solution were gently digested in the C tube (Miltenyi Biotec, Germany). The Gentle MACS tissue dissociator was running the h_tumor_02 program (Miltenyi Biotec, Germany). To prepare the single-cell suspension of tissue, the mixed-enzyme solution was pre-warmed in a 37°C hybridization incubator for 30min. The enzymatic digestion was stopped by diluting the enzyme solution with a medium containing penicillin-streptomycin and FBS. The solution was filtered through a 70μm Strainer filter (Life Technologies, USA). The solution was overlaid with the Ficoll-Paque plus (GE Healthcare, Sweden) using a serological pipette at the lowest speed setting. The centrifuge was done for the sample in the desired swing-out rotor at 400 x g and 20 °C for 30min. Using an Eppendorf pipette, the entire lymphocyte layer was aspirated, transferred to a fresh tube, and washed. Cell freezing medium was added to resuspend the single cells pellet and stored in liquid nitrogen. Each cell suspension was subjected to 5' single-cell RNA sequencing using Single Cell 5' Library and Gel Bead Kit V1, Single Cell A Chip Kit, and i7 Multiplex Kit (10x Genomics, USA) with a cell recovery target of 7000 except 5000, following the manufacturer's instructions. Gene expression libraries were sequenced on an Illumina HiSeq 2500, NovaSeq 6000, and HiSeq X Ten platforms.