GSM7634801: donor T1DAL_616755, alefacept treated, 104 wk post-treatment, responder (R), single cell TCR-seq; Homo sapiens; OTHER
PBMCs were stained with a flow cytometry panel (Figure S12) and sorted into non-naive CD8+ T cells (singlet, live, CD14- CD19- CD56- CD3+ CD8+ CD4-, not CD45RA+ CCR7+) from 12 donors (6 alefacept responders, 6 non-responders, as defined by preservation of C-peptide levels11,58) with new onset T1D at 104 wk (Visit 30) post-treatment with alefacept (T1DAL Trial, ITN) using the FACS Aria Fusion (Becton Dickinson) cell sorter. Cell viability and purity were >90%. Samples were sorted into RPMI with 2% human serum prior to single cell RNA-seq. A single cell suspension was prepared from sorted cells and loaded onto the 10x Chromium Controller (10X Genomics) according to the manufacturer's protocol, with a target capture of 5,000 cells per channel. Sequencing libraries were generated using the NextGEM Single Cell 5' v1.1 kit (10x Genomics). Three pools of gene expression and TCR libraries, each from two responders and two non-responders, were generated, with a ratio of 4:1 GEX:TCR. Each pool was run on a NextSeq P2 flowcell on a NextSeq 2000 sequencer (Illumina), with a target depth of 20,000 reads/cell for GEX and 5000 reads/cell for TCR libraries.