{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Giulia Picco"],"organism":["Mus musculus"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-15595"],"description":["Haematopoietic 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.  Here we performed RNA sequencing at multiple stages of EB differentiation to identify genes potentially important for mesoderm specification towards the haematoendothelial lineages.  Mouse ESCs were differentiated to embryoid bodies for 4 days, dissociated, stained for the Flk-1 and Pdgfra markers and Flk-1⁺/Pdgfrα⁻ and Flk-1⁺/Pdgfrα⁺ populations were sorted by FACS. RNA sequencing was performed at multiple stages of EB differentiation: day 0 (D0), day 2 (D2), day 4 (D4) and the two sorted populations (D4 Flk-1⁺/Pdgfrα⁻ and D4  Flk-1⁺/Pdgfrα⁺)."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Nucleic 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 D2 and 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.   For D4 sorted samples, 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.","Sample Collection - Mouse embryonic stem cells at day 0 were detached from the plates and resuspended to a single cell suspension using Trypsin EDTA. For the depletion of MEFs (mouse embryonic fibroblasts), the cell suspension was transferred to 0.1% Gelatin-coated plates with an excessive volume of ESC medium and incubated for 20-30 minutes to allow MEFs to loosely attached to the plates, followed by carefully harvesting the ESC-containing supernatant. Cells were strained with a 40 µM cell strainer (Greiner #542040), washed three times and counted in Trypan blue using a Countess II Automated Cell Counter."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","Processed Data","MAGE-TAB Files"],"data_protocol":["Data 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."],"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["Illumina HiSeq 2500"],"study_type":["RNA-seq of coding RNA"],"species":["Mus musculus"],"pubmed_authors":["Giulia Picco"],"additional_accession":[]},"is_claimable":false,"name":"RNA-seq of mouse Embryonic stem cells at different stages of embryoid body differentiation","description":"Haematopoietic 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.  Here we performed RNA sequencing at multiple stages of EB differentiation to identify genes potentially important for mesoderm specification towards the haematoendothelial lineages.  Mouse ESCs were differentiated to embryoid bodies for 4 days, dissociated, stained for the Flk-1 and Pdgfra markers and Flk-1⁺/Pdgfrα⁻ and Flk-1⁺/Pdgfrα⁺ populations were sorted by FACS. RNA sequencing was performed at multiple stages of EB differentiation: day 0 (D0), day 2 (D2), day 4 (D4) and the two sorted populations (D4 Flk-1⁺/Pdgfrα⁻ and D4  Flk-1⁺/Pdgfrα⁺).","dates":{"release":"2026-01-14T00:00:00Z","modification":"2026-05-27T00:09:08.943Z","creation":"2025-09-12T13:09:09.076Z"},"accession":"E-MTAB-15595","cross_references":{"ENA":["ERP180022"],"EFO":["EFO_0002944","EFO_0004170","EFO_0005518","EFO_0003816","EFO_0003738","EFO_0004184"]}}