biostudies-arrayexpressGiulia PiccoMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15596Haematopoietic and endothelial cells derive from a subset of Flk-1 expressing cells. In vitro differentiation of mouse embryonic stem cells (mESCs) to embryoid bodies (EBs) recapitulate several aspects of early embryonic development, including the formation of a subset of Flk-1 expressing cells. By day 4 of EB differentiation, mesodermal lineages with distinct lineage potentials can be identified based on the expression of the two surface markers Flk-1 and Pdgfra. Cells expressing Flk-1 alone (Flk-1⁺/Pdgfrα⁻) are enriched for haematoendothelial precursors, whereas double-positive cells (Flk-1⁺/Pdgfrα⁺) are enriched for primitive/cardiac mesodermal lineages. We previously identified six uncharacterized genes (Riken) as potential regulators of haematoendothelial or cardiac lineages commitment, namely I830077J02Rik,C130074G19Rik,1500009L16Rik,D630003M21Rik,D430041D05Rik and A530016L24Rik. We generated homozygous knockouts (KOs) of each of these 6 genes and performed bulk RNA sequencing of WT cells and gene KOs is the two sorted populations (Flk-1⁺/Pdgfrα⁻ and Flk-1⁺/Pdgfrα+) at day 4 of embryoid body differentiation.biostudies-arrayexpressNucleic Acid Extraction - Total RNA of WT and KO lines was extracted using ReliaPrep RNA Cell MiniPrep System (Promega, #Z6012) according to the manufacturer’s instructions.Sequencing - The sequencing libraries were loaded on an Illumina HiSeq2500 sequencer according to the Illumina TruSeq Rapid v2 protocol, with a single-read 50 bp setting.Library Construction - RNA-seq libraries were prepared according to Illumina TruSeq stranded mRNA protocol following manufacturer’s instructions, starting with 200 ng of total RNA.Sample Collection - Embryoid bodies at D4 were harvested by spinning at 300 rpm for 5 min, washed twice with PBS and dissociated using StemPro Accutase (Invitrogen, #A1110501). After two washes using PBS supplemented with 10% of FBS, single cells were filtered through a 0.22 µM cell strainer and counted for downstream applications. D4 dissociated EBs were incubated with 0.5 µg of Biotin anti-mouse CD309 (VEGFR2, Flk-1) antibody (Biolegend, #121904) and 1 µg of PE anti-mouse CD140a Antibody (Biolegend, #135905) for 1 X 106 cells in 100 µl of staining buffer (10% FBS in PBS). After an incubation of 30 minutes at 4°C and 3 washes with the staining buffer, 1 X 106 cells in 100 µl of staining buffer were incubated with the secondary antibody APC Streptavidin (Biolegend, 405207) for 15 minutes at 4°C, washed three times and resuspended in staining buffer. Cell sorting was performed using the BD FACSAria™ III Cell Sorter (BD Biosciences) and FACSAria™ II SORP.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Illumina adapters and poly-A sequences were trimmed off the reads, which were aligned to the GRCm38 reference genome using HiSat2 (v2.1.0). If a sample was sequenced on more than one flowcell, files were mapped independently, then sorted and merged using SAMtools. After sorting the alignment on read-name, quantification was performed using HT-seq count (v 0.11.2). Non-CCDS genes and transcripts were removed and only CCDS and miRbase transcript were used in the annotation. Ensembl v96 was used for transcript/gene annotation.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina HiSeq 2500RNA-seq of coding RNAMus musculusGiulia PiccofalseRNA-seq of mouse wild-type and I830077J02Rik,C130074G19Rik,1500009L16Rik,D630003M21Rik,D430041D05Rik and A530016L24Rik gene knockouts in Flk-1+/Pdgfra+ and Flk-1+/Pdgfra- sorted populations at day 4 of embryoid body differentiationHaematopoietic and endothelial cells derive from a subset of Flk-1 expressing cells. In vitro differentiation of mouse embryonic stem cells (mESCs) to embryoid bodies (EBs) recapitulate several aspects of early embryonic development, including the formation of a subset of Flk-1 expressing cells. By day 4 of EB differentiation, mesodermal lineages with distinct lineage potentials can be identified based on the expression of the two surface markers Flk-1 and Pdgfra. Cells expressing Flk-1 alone (Flk-1⁺/Pdgfrα⁻) are enriched for haematoendothelial precursors, whereas double-positive cells (Flk-1⁺/Pdgfrα⁺) are enriched for primitive/cardiac mesodermal lineages. We previously identified six uncharacterized genes (Riken) as potential regulators of haematoendothelial or cardiac lineages commitment, namely I830077J02Rik,C130074G19Rik,1500009L16Rik,D630003M21Rik,D430041D05Rik and A530016L24Rik. We generated homozygous knockouts (KOs) of each of these 6 genes and performed bulk RNA sequencing of WT cells and gene KOs is the two sorted populations (Flk-1⁺/Pdgfrα⁻ and Flk-1⁺/Pdgfrα+) at day 4 of embryoid body differentiation.2026-01-14T00:00:00Z2026-05-26T15:02:37.171Z2025-09-12T13:21:21.434ZE-MTAB-15596ERP180024EFO_0002944EFO_0004170EFO_0005518EFO_0003816EFO_0003738EFO_0004184