{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Nadezhda Azbukina"],"organism":["Homo sapiens"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-15826"],"description":["We used a pooled, multiplexed CRISPR/Cas9 gene knock-out (KO) experiment with single-cell transcriptomic readout to perturb and validate selected TF regulomes . We designed gRNAs and generated a pooled lentiviral library targeting 12 TFs with specific midbrain or hindbrain expression or high regulatory centrality. IPSCs carrying a doxycycline-inducible Cas9 cassette in the AAVS1 safe harbor locus were transduced, mosaic organoids were generated, and perturbations were induced at neuroepithelium stage from day 7 to 14. Mosaic organoids were analyzed using scRNA-seq and gRNA amplicon sequencing at day 30 and day 70, recovering 31,857 cells, among which a gRNA was detected in 8711 cells."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Library Construction - Cells positive for fluorescent reporters were selected using FACS and these cells were then loaded onto the 10x Chromium using the Chromium Next GEM Single Cell 5’ v2 Dual Index kit (10x Genomics, PN-1000265) targeting 10,000 cells per reaction with two reactions per time point. Single-cell transcriptome and CRISPR gRNA libraries were prepared following manufacturer protocols.","Sample Collection - Five organoids were pooled and dissociated into single cell suspensions at day 30 and day 70. Cells positive for fluorescent reporters were selected using FACS and these cells were then loaded onto the 10x Chromium using the Chromium Next GEM Single Cell 5’ v2 Dual Index kit (10x Genomics, PN-1000265) targeting 10,000 cells per reaction with two reactions per time point.","Nucleic Acid Extraction - Cells positive for fluorescent reporters were selected using FACS and these cells were then loaded onto the 10x Chromium using the Chromium Next GEM Single Cell 5’ v2 Dual Index kit (10x Genomics, PN-1000265) targeting 10,000 cells per reaction with two reactions per time point. Single-cell transcriptome and CRISPR gRNA libraries were prepared following manufacturer protocols.","Sequencing - The transcriptome libraries and gRNA libraries were pooled and sequenced on the NovaSeq 6000 platform using 26/10/10/90 as cycle parameters.","Growth Protocol - The final mixed pool of cells was used to generate Qian organoids following the previously described method (Qian Reference). To induce Cas9 expression and therefore create knockouts in targeted genes, 2 μg/ml doxycycline (Clontech, 631311) was added with each media change starting on day 7 until day 14. Organoids were returned to their normal media on day 15. Fluorescence was monitored throughout culturing to ensure KOs reporters were not silenced throughout differentiation."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","Processed Data","MAGE-TAB Files"],"data_protocol":["Data Transformation - We used Cell Ranger (v.7.0.1) using flag multi to obtain count matrices of transcriptome and gRNAs. We used human transcriptome (hg38, provided by 10x Genomics), and a table of guide sequences for GEX and gRNA reads mapping respectively. To facilitate efficient gRNA detection, the pattern and sequence entries in the feature reference files were adjusted to 12bp instead of 20bp. Then, as with the time course, count matrices were further processed using the Seurat R package. Cells were filtered on the basis of the number of counts (>600,<20,000 for day 30) or detected genes (>500,<7500 for day 70) and the fraction of mitochondrial genes (<0.15 for day 30 and <0.1 for day 70). Transcript counts were normalized to the total number of counts for that cell, multiplied by a scaling factor of 10,000 and subsequently natural-log transformed (NormalizeData()). 10X lanes from the same time points were integrated using CSS method as for the time course data. Datasets were annotated combining label transfer from the time course dataset (using css_project() function of CSS package) and marker gene expression. To assign gRNA labels, the 10x gRNA calling outputs (protospacer_calls_per_cell.csv) were first filtered to retain cells with gRNAs targeting a single gene. For cells with gRNAs targeting multiple genes, additional filtering was applied to identify the best call based on z-scaled UMI > 5 and a total UMI count > 10. Cells lacking gRNAs calls were excluded from further analysis. Cells with effective perturbations were identified using Mixscape68 as implemented in Seurat using cells with all other gRNAs as a control. Regulon activities upon CRISPR-based knockout were quantified using Seurat function AddModuleScore() considering the positive and negative regulons of TF of interest as inferred in Pando. To visualize the detected gRNAs and regulon activities on the UMAP embeddings, their densities were estimated using Nebulosa69 (weight=1 for gRNA). Differentially expressed genes (DEGs) were identified for each cluster with FindMarkers() function in the seurat package using wilcoxon test (log2 fold change >0.5, adjusted p<0.05, detection rate>5%, detection rate difference>10%) and analyzed for gene ontology enrichment using clusterProfiler."],"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["Illumina NovaSeq 6000"],"study_type":["RNA-seq of coding RNA from single cells"],"species":["Homo sapiens"],"pubmed_title":["Multi-omic human neural organoid cell atlas of the posterior brain"],"pubmed_authors":["Hsiu-Chuan Lin","Zhisong He","Nadezhda Azbukina, Zhisong He, Hsiu-Chuan Lin, Malgorzata Santel, Bijan Kashanian , Ashley Maynard, Tivadar Török, Ryoko Okamoto, Marina Nikolova, Sabina Kanton, Valentin Brösamle, Rene Holtackers, J. Gray Camp, Barbara Treutlein","Barbara Treutlein","Nadezhda Azbukina"],"additional_accession":[]},"is_claimable":false,"name":"scRNA-seq CRISPR screening of human posterior brain organoids","description":"We used a pooled, multiplexed CRISPR/Cas9 gene knock-out (KO) experiment with single-cell transcriptomic readout to perturb and validate selected TF regulomes . We designed gRNAs and generated a pooled lentiviral library targeting 12 TFs with specific midbrain or hindbrain expression or high regulatory centrality. IPSCs carrying a doxycycline-inducible Cas9 cassette in the AAVS1 safe harbor locus were transduced, mosaic organoids were generated, and perturbations were induced at neuroepithelium stage from day 7 to 14. Mosaic organoids were analyzed using scRNA-seq and gRNA amplicon sequencing at day 30 and day 70, recovering 31,857 cells, among which a gRNA was detected in 8711 cells.","dates":{"release":"2026-03-17T00:00:00Z","modification":"2026-03-17T02:03:49.32Z","creation":"2025-10-23T12:15:55.169Z"},"accession":"E-MTAB-15826","cross_references":{"ENA":["ERP182815"],"EFO":["EFO_0002944","EFO_0004170","EFO_0003789","EFO_0005684","EFO_0005518","EFO_0003816","EFO_0004184"],"doi":["10.1101/2025.03.20.644368"]}}