biostudies-arrayexpressJasmin StraubeHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15981Myeloproliferative neoplasm (MPN) or post-MPN acute myeloid leukemia patient bone marrow aspirates were sorted for CD34+ hematopoietic stem and progenitor cells (HSPCs), followed by 10x 3' single cell RNA sequencing and long read sequencing to study the consequences of mutation combination on HSPCs.biostudies-arrayexpressLibrary Construction - We performed single cell capture and RNA-seq library preparation according to the 10x Genomics Chromium 3’ v3.1 User Guide (https://cdn.10xgenomics.com/image/upload/v1660261285/support-documents/CG000204_ChromiumNextGEMSingleCell3_v3.1_Rev_D.pdf).Nucleic Acid Extraction - We performed single cell capture and RNA-seq library preparation according to the 10x Genomics Chromium 3’ v3.1 User Guide (https://cdn.10xgenomics.com/image/upload/v1660261285/support-documents/CG000204_ChromiumNextGEMSingleCell3_v3.1_Rev_D.pdf).Sample Collection - Myeloproliferative neoplasm (MPN) or post-MPN acute myeloid leukemia (AML) patient bone marrow samples were stained with CD3-PE/Cy7, CD34-PE, CD14-FITC and CD15-APC . CD34+ Hematopoietic stem and progenitor cells (HSPCs) were FACS sorted on the BD FACSAria III (BD Sciences).Sequencing - Sequencing was performed on an Illumina NextSeq500 according to required sequencing reads, using 100 cycle kit in the following configuration: Read1 – 28 bases, Index1 – 10 bases, Index2 – 10 bases, Read2 – 90 bases.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - The here uploaded data are raw data and are not normalized or transformed.Sequence Alignment - MPN and post-MPN AML sequenced reads were adapter trimmed and processed through cellranger (v6.0.1, or MPN10 v3.0.2) with GRCh38 genome build to obtain a counts per single cell and gene matrix.MetabolomicsUnknownTranscriptomicsGenomicsProteomics10x Genomics ChromiumHPCFACSAria III (BD Sciences)NextSeq 500Myeloproliferative neoplasms (MPNs) are caused by acquired mutations in hematopoietic stem and progenitor cells (HSPCs). The acquisition of additional mutations like TP53 and the overall mutational burden influence a patient’s risk of disease progression toward lethal post-MPN acute myeloid leukemia (AML). Recent technological advancements in linking single-cell gene expression with genotype have improved our understanding of tumor heterogeneity. However, current methodologies have limitations in simultaneously genotyping low-expression genes (such as JAK2 ) alongside other pathogenic loci. To address this, we developed a novel lo ng read genotyping pipeline of cDNA tr anscripts called LOTR-Seq, which can genotype the full length of expressed transcripts of 30 genes at once. Using LOTR-Seq, we genotyped HSPCs at the JAK2 V617 locus in 9,075 single cells from eight patients with chronic phase MPN (CP-MPN) and in 5,016 cells from four patients with post-MPN AML. We then linked the mutations to the single cell transcriptome of 29,712 JAK2 V617F-driven CP-MPN cells and 16,895 post-MPN AML cells. In our analysis of post-MPN AMLs, we identified nine mutated loci across six genes ( JAK2, IDH1/2, TP53, SRSF2, U2AF1 ) and linked these mutations to specific transcriptional phenotypes. Overall, LOTR-Seq provides novel insights into the evolution of post-MPN AML.RNA-seq of coding RNA from single cellsHomo sapiensSingle cell long-read genotyping of transcripts reveals discrete mechanisms of clonal evolution in post-myeloproliferative neoplasm acute myeloid leukemiaJulian Grabek, Jasmin Straube, Leanne Cooper, Rohit Haldar, Ranran Zhang, Inken Dulige, Matthew Barker, Will Gatehouse, Helen Christensen, Gerlinda Amor, Victoria Y. Ling, Caroline McNamara, David M. Ross, Andrew Perkins, Megan J. Bywater, Steven W. LaneJasmin StraubefalseSingle cell long read genotyping of transcripts reveals discrete mechanisms of clonal evolution in post-MPN AMLMyeloproliferative neoplasm (MPN) or post-MPN acute myeloid leukemia patient bone marrow aspirates were sorted for CD34+ hematopoietic stem and progenitor cells (HSPCs), followed by 10x 3' single cell RNA sequencing and long read sequencing to study the consequences of mutation combination on HSPCs.2026-06-25T00:00:00Z2026-06-25T01:01:06.186Z2025-11-03T17:01:28.237ZE-MTAB-15981ERP183612EFO_0002944EFO_0004170EFO_0005684EFO_0004917EFO_0005518EFO_0003816EFO_000418410.1101/2025.08.18.670417