GSM7719629: ovCHA018, scTCR-seq; Homo sapiens; RNA-Seq
Tumor tissues were minced and dissociated using the enzyme mix from Tumor Dissociation Kit, human (130-095-929, Miltenyi Biotec) with the h_tumor_02 program twice on the gentleMACS dissociator/C tube (130-093-237, Miltenyi Biotec). At the end of each program, the sample was incubated in a pre-warmed 37°C incubator for 30 minutes. The reaction was stopped with a medium containing penicillin-streptomycin and FBS. Then, the sample was filtered through a 70-μm strainer filter (352350, Life Technologies) and processed with Ficoll-Paque PLUS (17-1440-02, GE Healthcare) to remove dead cells. Cell pellets were washed and resuspended in a cell-freezing medium and stored in liquid nitrogen. Single cell suspensions were subjected to 5' single-cell RNA sequencing using the Single Cell 5′ Library and Gel Bead Kit V1, Single Cell A Chip Kit, i7 Multiplex Kit (10x Genomics) according to the manufacturer's protocol. Library for T92 was constructed using the Chromium Next GEM Single Cell 5' Library & Gel Bead Kit v1.1, and Chromium Next GEM Chip G Single Cell Kit (10x Genomics) instead. Each target for cell recovery was set at 5,000-7,000 cells. TCR-enriched and BCR-enriched libraries were prepared using the Single Cell V(D)J Enrichment Kit, Human T Cell, and Single Cell V(D)J Enrichment Kit, Human B Cell (10× Genomics) according to the manufacturer's protocol.