GSM5502407: STIM_24h_CAR_PacBio; Homo sapiens; RNA-Seq
Eight anti-GPC2 and six anti-CD276 CAR T- cells were first thawed separately and recover for overnight. Then they were pooled the same way according to its counterpart experiment co-cultured with tumor cells using an E:T ratio of 1:1 for individual anti-GPC2 CAR T- cells expect G27; and 1:4 for each anti-CD276 and G27 CAR T- cells. Pooled CAR T-cells were evenly divided into seven fractions for multimodal single cell assay. One of the 7 fractions were taken as CTRL_0h and remaining 6 fractions were split into two groups: one group of 3 fractions cultured without target tumor cells for 24 hours (CTRL_24h) and other group of 3 fractions co-cultured with target tumor cells for 24 hours (STIM_24h). To improve the cell viability to >85%, dead cells were removed by magnetic negative selection (Dead Cell Removal Kit, Miltenyi Biotec, Cat# 130-090-101). For 2 groups of pooled CAR T-cells at 24 hours, Totalseq-C human 'hashtag' antibodies were used for each of CAR T-cell triplicates in 2 groups allowing identification of different samples in the analysis, and then triplicates in each group were combined. Next, three CAR T- cell pools (CTRL_0h, CTRL_24h, and STIM_24h) were stained with a cocktail of TotalSeq-C human lyophilized panel (BioLegend) of 31 surface proteins. After washed three times, CAR-T cells were resuspended in PBS, and counted before proceeding immediately to the single cell immune profiling using a 10X Genomics Chromium system (10x Genomics, Pleasanton, CA). Briefly, CAR T-cells were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5' Chromium Single Cell Immune Profiling Next GEM v1.1 chemistry. For detection of 14 CAR binders, a fraction of amplified full-length cDNA was used for PCR with CAR binder specific reverse primers, then size selected products (>600bp) were sequenced by PacBio Sequel II for full-length of binder sequences.