biostudies-arrayexpressJan KubovčiakMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15355To investigate the biological role of the germline JAK2-R1063H mutation, which may contribute to susceptibility and pathogenesis of myeloproliferative neoplasms, we generated a mouse model by introducing the R1063H variant into the endogenous Jak2 locus using CRISPR/Cas9 genome editing. This model allowed us to explore not only hematopoietic but also non-hematopoietic compartments potentially affected by the mutation. Given the ongoing debate regarding the presence and functional impact of somatic JAK2-V617F in endothelial cells — and their possible involvement in thrombotic complications — we specifically examined whether Jak2-R1063H in endothelial cells drives similar pathogenic phenotype. Therefore, we isolated lung endothelial cells (CD45⁻/CD31⁺) from 12- and 18-month-old Jak2-R1063H and wild-type mice and conducted gene expression profiling.biostudies-arrayexpressLibrary Construction - Libraries were prepared using Smarter stranded total RNA-Seq kit v2 - pico Input mammalian PN-634413 (Takara).Sample Collection - Lungs were divided into three portions, each incubated in serum-free medium with dispase (1:300), collagenase I (2 mg/mL), and DNase I (1:1000). Tissue was finely minced, brought to 1 mL with enzyme mix, and digested at 37°C with shaking (800 rpm) in three 10-minute cycles, followed by a final 20-minute digestion. Digests were pooled into cold medium with 10% FBS to stop enzyme activity, filtered through a 70 µm mesh, and centrifuged (500 × g, 10 min, 4°C). Pellets were treated with ACK lysis buffer to remove erythrocytes, incubated on ice for 5 minutes, washed with medium containing 10% FBS, and centrifuged again (500 × g, 5 min, 4°C). The cell pellet was resuspended in 10% FBS medium containing fluorescently conjugated antibodies: anti-mouse CD45-Pacific Blue (PB; Biolegend), CD31-PE (eBioscience), and EpCAM-APC (CD236; eBioscience), each at a final dilution of 1:400. Unstained and single-color controls were included. Cells were incubated on ice for 15–30 minutes. Following staining, cells were washed with 10 mL of 10% FBS medium and centrifuged again (500 × g, 5 minutes, 4°C). The pellet was resuspended in 200–500 µL of HBSS with 3% FBS. The CD31⁺ population was FACS sorted directly into RLT lysis buffer; EpCAM⁺ cells served as epithelial controls.Sequencing - Libraries were sequenced on Illumina NextSeq 2000 instrument using P3 XLEAP-SBS Reagent Kit (100 Cycles) with 122 nt single-end read length configuration.Nucleic Acid Extraction - Total RNA was isolated using micro kit (Qiagen) from 50 000 FACS-sorted cells.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Sequencing reads were mapped with STAR v2.7.9a and expression on gene level was quantified from mapping reads with salmon v1.10.1. Estimated gene-level counts are provided as processed files.Sequence Alignment - Sequencing reads were processed using the bioinformatic pipeline nf-core/rnaseq v3.12.0. Individual steps included removing sequencing adaptors and low-quality reads with Trim Galore v0.6.7 and cutadapt v3.4, mapping to reference genome GRCm39 (Ensembl annotation version 112) with STAR v2.7.9a and quantifying expression on gene level with salmon v1.10.1.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsNextSeq 2000RNA-seq of coding RNAMus musculusJan KubovčiakLucie LáníkováfalseExpression profiling of endothelial cells from old (12 months) and very old (18 months) homozygous Jak2 R1063H mice and wild-type controlsTo investigate the biological role of the germline JAK2-R1063H mutation, which may contribute to susceptibility and pathogenesis of myeloproliferative neoplasms, we generated a mouse model by introducing the R1063H variant into the endogenous Jak2 locus using CRISPR/Cas9 genome editing. This model allowed us to explore not only hematopoietic but also non-hematopoietic compartments potentially affected by the mutation. Given the ongoing debate regarding the presence and functional impact of somatic JAK2-V617F in endothelial cells — and their possible involvement in thrombotic complications — we specifically examined whether Jak2-R1063H in endothelial cells drives similar pathogenic phenotype. Therefore, we isolated lung endothelial cells (CD45⁻/CD31⁺) from 12- and 18-month-old Jak2-R1063H and wild-type mice and conducted gene expression profiling.2025-08-22T00:00:00Z2025-08-18T10:45:01.94Z2025-07-14T13:11:39.554ZE-MTAB-15355ERP176753E-MTAB-14686EFO_0002944EFO_0004170EFO_0004917EFO_0005518EFO_0003816EFO_0003738EFO_0004184