{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Pietro Zoppoli"],"organism":["Mus musculus"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-14503"],"description":["Here we use mouse gastruloids to investigate the role of an ultraconserved lncRNA, T-UCstem1, in the formation of the mammalian body plan using single cells transcriptomics, we provide unprecedented evidence that T-UCstem1 is a key regulator of gastruloid development and is required for the correct specification of the anterior-posterior axis. Specifically, knock down of T-UCstem1 results in aberrant gastruloid development, which is characterized by altered spatiotemporal expression of the differentiation markers and persistence of pluripotency genes. Single cell analysis reveals higher cellular heterogeneity in T-UCstem1 KD gastruloids. Notably, the presence of cell populations characterized by the co-expression of pluripotency and differentiation markers points to a key role of T-UCstem1 in establishment and maintenance of proper cellular identity."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Library Construction - After GEM generation, all the steps of cDNA synthesis, clean-up and amplification, DNA fragmentation, end-repair, adapter ligation and indexing PCR-amplification were done with the Chromium Single Cell 3‘ reagents following the manufacturer’s protocol while the DNA purification as well as the size selection steps were done with SPRIselect magnetic beads (Beckman Coulter, Brea, CA) as indicated by the 10X protocol. Finally, the concentration of each indexed library was determined by using Qubit dsDNA BR Assay (Thermo Fisher Scientific, USA) and their quality were assessed with the D1000 screentape on the TapeStation 4150 (Agilent Technologies, Santa Clara, CA).","Sequencing - An equimolar amount of both the DNA libraries was pooled together and subject to cluster generation and sequencing into the Illumina NextSeq550 System (Illumina, San Diego, CA) in a paired-end dual index format with thefollowing sequencing cycles: 28 for Read1, 10 for Index i7, 10 for Index i5 and 90 for Read2.","Sample Collection - Gastruloid formation assay was performed as described (Baillie-Johnson et al., 2015; Cermola et al., 2021). Naïve ESCs were cultured at low density (250 cells/cm2) and the resulting colonies were dissociated with accutase (Sigma-Aldrich; cat: A6964; 3 min at 37°C). Cells were seeded in N2B27 at 1.0-2.5 x 102 cells/well (40 μL) in ultra-low attachment 96 multi-well plates (Corning Costar; cat: 7007) and allowed to aggregate. At 48 h AA, CHIR was added (3 μM) to the culture medium and maintained for 24 h. From 72 h onward, the medium (150 μL) was refreshed daily up to 120 h. DKK-1 recombinant protein (200 ng/mL) and WAY262611 (0.1, 0.20, 0.25, 0.50, 0.75 and 1μM) were added during throughout the assay. Gastruloids from NT and T-UCstem1 KD-1 were collected after 120 h.","Nucleic Acid Extraction - gastruloids were collected in 15 mL tubes, pelleted by gravity and dissociated by trypsin incubation at 37 ̊ for 7 min, washed with PBS and resuspended in cold PBS supplemented with 0.1% BSA. The single-cell suspensions were then visually inspected for determining cell number and viability as well as the absence of residual cellular aggregates and finally loaded into the Chromium Controller (10X Genomics) to generate single cell GEMs."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","Processed Data","MAGE-TAB Files"],"data_protocol":["Data Transformation - The Cell Ranger Single-Cell software (v.8.0, 10x Genomics) was used for processing data as according to manufacturer. Briefly, reads will be first assigned to cells and then aligned to the mouse genome (refdata-gex-GRCm39-2024-A, GRCm39) using STAR(Dobin et al., 2013)."],"omics_type":["Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["NextSeq 550"],"study_type":["RNA-seq of coding RNA from single cells"],"species":["Mus musculus"],"pubmed_authors":["Pietro Zoppoli"],"additional_accession":[]},"is_claimable":false,"name":"Non-cell-autonomous control of gastruloid development by the lncRNA T-UCstem1 2 through DKK1-dependent modulation of WNT signalling","description":"Here we use mouse gastruloids to investigate the role of an ultraconserved lncRNA, T-UCstem1, in the formation of the mammalian body plan using single cells transcriptomics, we provide unprecedented evidence that T-UCstem1 is a key regulator of gastruloid development and is required for the correct specification of the anterior-posterior axis. Specifically, knock down of T-UCstem1 results in aberrant gastruloid development, which is characterized by altered spatiotemporal expression of the differentiation markers and persistence of pluripotency genes. Single cell analysis reveals higher cellular heterogeneity in T-UCstem1 KD gastruloids. Notably, the presence of cell populations characterized by the co-expression of pluripotency and differentiation markers points to a key role of T-UCstem1 in establishment and maintenance of proper cellular identity.","dates":{"release":"2025-09-30T00:00:00Z","modification":"2025-09-30T01:04:50.878Z","creation":"2024-09-27T12:58:24.593Z"},"accession":"E-MTAB-14503","cross_references":{"ENA":["ERP164604"],"EFO":["EFO_0002944","EFO_0004170","EFO_0005684","EFO_0005518","EFO_0003816","EFO_0004184"]}}