GSE169603
Splicing patterns in SF3B1 mutated uveal melanoma generate shared immunogenic tumor-specific neo-epitopes [scTCR-seq]
Purpose: In this study, we show that mutations of the splicing factor SF3B1 in uveal melanoma (UM) generate immunogenic neo-antigens. Overall design: Thawed  PBMC  from  patient  UM1 were  stained  with PE  and  APC  tetramers  loaded with  peptide 37,  then  tetramer  positive  cells  were  positively enriched  using  anti-APC  and  anti-PE  microbeads (Miltenyi Biotech), stained with CD3 A700 and CD8 FITC and finally with DAPI. The positive fraction was sorted in a FACS ARIA (BD). To combine 5 tetramer positive populations from 2 donors (14, 17, 26  and  37  for  UM2  and  18  for  UM3)  the  tetramers  were  prepared  using  5  different  TotalSeq-PE streptavidins  (BioLegend)  and  1  classical  fluorochrome-streptavidin  (APC,  PE-CF594,  PE-Cy5,  PE-Cy7), PE-CF594 was used for UM3 who had only one population sorted. The PBMC were stained with the 4 pairs of tetramers for patient UM2 and 1 pair of tetramers for patient UM3, enriched with anti PE-microbeads (Milteny Biotech), stained with CD3 A700 and CD8 FITC and DAPI and sorted separately. The  cells  were  then  counted,  mixed  and  loaded  onto  a  Chromium  controller  using  Chromium  next GEM  Single  Cell  V(D)J  reagent  kit  with  feature  barcoding  technology  according to manufacturer's instructions