biostudies-arrayexpressBaptiste AlbertiDrosophila melanogasterCellRangerhttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-14447Single-cell RNA sequencing (scRNA-seq) in Drosophila embryos at 2.5 to 3.5 hours after egg lay (corresponding to developmental stages 5 to 7, with a majority of stage 6). The experiments were performed in the nos-phiC31; attP40 fly line containing a reporter construct with the sequence of a candidate enhancer driving the expression of the CD2 reporter gene. scRNA-seq libraries were generated using the 10X 3' end v3.1 kit, with three batches of embryo collection, dissociation, 10X chromium encapsulation, and sequencing performed from the same flies.biostudies-arrayexpressSample Collection - Freshly hatched adults from the pool of transgenic flies containing our library of candidate enhancers were combined in 5 embryo collection vials with standard apple cap plates. After three 45-minute pre-lays, Drosophila embryos were collected on apple juice plates for one hour and incubated for another 2.5 hours at 25°C. We verified that the collected embryos mostly span developmental stages 5 to 7. The embryos were dechorionated using 2,6% bleach for 2 minutes, washed with water and PBS + 0.1% Triton X-100, and finally resuspended in 1 ml of ice-cold PBS + 0.1% Triton X-100 and kept on ice. Embryos were washed with ice-cold PBS, resuspended in 10 mL of dissociation buffer (PBS 1X + 0.1% BSA), and dissociated on ice in a Dounce homogenizer with gentle strokes of the loose pestle. This process was repeated for all collected embryos, using a small number of embryos each time. The cell suspension was transferred to 15 mL Falcon tubes and centrifuged for 10 minutes at 40g at 4°C to pellet debris. Cells in the supernatant were transferred to new Falcon tubes and centrifuged again for 10 minutes at 800g at 4°C. The supernatant was discarded, and cell pellets were combined and resuspended in 0.5 mL of dissociation buffer. Two additional rounds of centrifugations, each for 5 minutes at 800g at 4°C were performed to remove as much debris as possible. Cells were counted using a Malassez counting chamber and the concentration was adjusted to 900 cells/µl in the dissociation buffer. Single cells were encapsulated into droplets in the Chromium Controller instrument for cell lysis and barcoded reverse transcription of mRNA. 40 µl of cDNA were recovered, and 10 µl (25%) were used for amplification, fragmentation, and Illumina library construction.Library Construction - Library preparation was performed with the 10X library kit provided with the Chromium Next GEM Single Cell 3ʹ Kit v3.1Nucleic Acid Extraction - Cells were encapsulated with the 10X chromium device. RNA extraction was performed following the10XGenomics Chromium Next GEM Single Cell 3ʹ Kit v3.1Sequencing - All three samples were sequenced with a NovaSeq (Illumina) sequencer, using 150-bp paired-end reads, yielding 500 million reads per libraryOrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Sequencing reads were controlled first by fastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc) to assure their good quality before mapping. They were aligned to the custom genome index using cellranger count (no citation, just mentioning the software plus version (see here: https://www.10xgenomics.com/support/software/cell-ranger/latest/miscellaneous/cr-citations)) with 14 processing cores. The 10X Genomics cell and UMI barcodes were isolated by selecting the first 28 nucleotides in R1 with the parameter r1-length set to 28. After mapping, 24,886, 23,125, and 23,437 cells were obtained for replicates 1 to 3, respectively. Each gene expression matrix went through stringent quality filtering. Non-viable cells, debris, or empty droplets were excluded if they contained fewer than 500 expressed genes and/or 2,000 UMIs. To prevent duplicates, a maximum threshold of 6,000 genes and/or 50,000 UMIs was set. Cells with more than 15% of mitochondrial genes or more than 36% of ribosomal genes were excluded. After quality control, 1,934, 1,963, and 2,691 cells were retained for each replicate. To merge all three replicates, the Seurat (https://doi.org/10.1016%2Fj.cell.2021.04.048) integration vignette was followed. Datasets were normalized separately, and highly variable genes were identified using the FindVariableFeatures function with the mvp method. Both FindIntegrationAnchors and IntegrateData functions were used to generate a single dataset of 6,588 cells. The standard Seurat pipeline was followed for analysis, including scaling, PCA, neighbor graph generation, clustering, and UMAP visualization.Sequence Alignment - Alignement was performed with CellRanger v 7.2.0 with a custom Drosophila melanogaster genome (dm6)MetabolomicsUnknownTranscriptomicsGenomicsProteomics10X Genomics Chromium ControllerIllumina NovaSeq XRNA-seq of coding RNA from single cellsDrosophila melanogasterIntegrating Single-Cell RNA Sequencing with Spatial Reconstruction to predict enhancer activity in early Drosophila developmentBaptiste AlbertiB. Alberti, S. Vincent, I. Stévant, D. Lajoignie, P. Villoutreix, Y. Ghavi-HelmYad Ghavi-HelmfalseSingle-Cell RNA sequencing of stage 6 Drosophila embryos with 25 enhancer reporter assaysSingle-cell RNA sequencing (scRNA-seq) in Drosophila embryos at 2.5 to 3.5 hours after egg lay (corresponding to developmental stages 5 to 7, with a majority of stage 6). The experiments were performed in the nos-phiC31; attP40 fly line containing a reporter construct with the sequence of a candidate enhancer driving the expression of the CD2 reporter gene. scRNA-seq libraries were generated using the 10X 3' end v3.1 kit, with three batches of embryo collection, dissociation, 10X chromium encapsulation, and sequencing performed from the same flies.2025-07-14T00:00:00Z2025-07-10T14:00:52.184Z2024-09-11T15:38:35.978ZE-MTAB-14447ERP164043E-MTAB-14445EFO_0002944EFO_0004170EFO_0005684EFO_0004917EFO_0005518EFO_0003816EFO_0004184