Hiseq next generation sequencing instrument

LSK (Lin⁻Sca1⁺c-kit⁺) cells were sorted from Jak2^VF/VF mice treated with vehicle, Palbociclib, Ruxolitinib or Palbociclib plus Ruxolitinib combination using a FACS Aria II (BD Biosciences). Total RNA was extracted from LSK cells using RNeasy Mini kit (Qiagen). RNA sequencing was performed using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB) and Hiseq next-generation sequencing instrument (Illumina). Jak2V617F LT-HSC microarray data accession number: GSE79198. MPN patient data accession number: GSE54644. RNA-seq data generated in this study are deposited to NCBI GEO database (GSE173814).

LSK cells from the BM of control and U2af1-deleted BMT mice, and lineage-negative (Lin^-) cells from the BM of control and primary U2af1-deleted mice were first enriched using Lineage cell depletion kit from Miltenyi and then sorted using a FACS Aria II at 7 days after pI-pC induction. Total RNA was extracted from BM LSK and Lin^- cells using RNeasy Mini kit (Qiagen). RNA sequencing was performed using Ultra II Directional RNA Library prep kit for Illumina (from NEB) and Hiseq next-generation sequencing instrument (Illumina). Gene set enrichment analysis was performed as described [19 ]. To identify splicing events, rMATS [20 ] was used. RT-PCR followed by gel electrophoresis was used to identify splicing events. Image J software was used to quantify the band intensity. Sequences of the primers are available in the supplemental Table 3. RNA-seq data from U2af1 cKO mice will be deposited to publicly available database (GEO). RNA-seq data for MP (Lin^-c-kit⁺) cells from U2af1 S34F knock-in mice: GSE112174.