Illumina NovaSeq 6000 paired end sequencing; scRNA-seq of PBMCs from stable ITP patients before TPO-RA discontinuation. STOPAGO study.
ScRNA-seq analyses were performed on frozen PBMCs isolated from heparin blood. Samples originated from 8 patients at baseline (4 with SCROT and 4 with SROT). PBMCs were thawed according to 10X Genomics protocol. Cells were counted, diluted at 1,000 cells/mL in PBS+0.04% and 20,000 cells were loaded in the 10x Chromium Controller to generate single-cell gel-beads in emulsion. After reverse transcription, gel-beads in emulsion were disrupted. Barcoded complementary DNA was isolated and amplified by PCR. Following fragmentation, end repair and A-tailing, sample indexes were added during index PCR.