Chromothripsis-associated chromosome 21 amplification orchestrates transformation to blast phase MPN through targetable overexpression of DYRK1A
To investigate the effect of chr21amp on cell differentiation, we performed droplet-based, high-throughput single-cell RNA-seq on Lin-CD34+ cells and total mononuclear cells. Overall design: We used the 10x genomics platform to profile single-cell transcriptomes of 9,000 CD34+ lineage negative and 9,000 viable mononuclear cells were sorted into 30uL PBS/0.05% BSA (non-acetylated) in a 1.5ml DNA lo-bind Eppendorf. Samples were processed according to the 10x protocol using the Chromium Single Cell 30 library and Gel Bead Kits v3.0 (10x Genomics). Cells and reagents were prepared and loaded onto the chip and into the Chromium Controller for droplet generation. Reverse transcription was conducted in the droplets and cDNA recovered through demulsification and bead purification. Pre-amplified cDNA was used for library preparation, multiplexed and sequenced on a Novaseq S4, aiming to obtain > 50,000 reads per cell. A preliminary, low-depth run was performed to more accurately estimate the number of cells and total sequencing required. BPMPN samples are denoted 'S' and healthy controls 'HC'. Please refer to the manuscript for downstream processing.