biostudies-arrayexpressNadezhda AzbukinaHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15622In order to systematically investigate, to which extent cell lines and experimental batches affect the consistency of organoid patterning, we conducted a multiplexed morphogen patterning experiment to study the reproducibility of the effects induced by retinoic acid, FGF-8, SHH and CHIR. We have tested influence of 2 neural induction methods, 2 experimental batches and 4 cell lines: H1 (XY), H9 (XX), WTC (XY) and WIBJ (XX) on cell type composition consistency after morphogen patterning using single-cell transcriptomic readout (Parse Bioscience) and pooling 3 organoids for each condition.biostudies-arrayexpressSample Collection - After d21 in culture 3 organoids per condition per cell line were dissociated individually using CyBio FeliX liquid handler robot with thermoshaker. For dissociation we used a papain-based dissociation kit (Miltenyi Neural dissociation kit). Each organoid was dissociated using 410uL of enzyme mix 1 and 6uL of enzyme mix 2. Then single cell suspension from the same condition were pooled together (H1 and H9; WTC and WIBJ2).Sequencing - Libraries were sequenced in S2 flowcell with Illumina Nova Seq technology, using paired-end 64/8/8/58 configuration.Nucleic Acid Extraction - Each individual single cell suspension has been followed by fixation and permeabilization procedures, which were performed according to the manufacturer specification (ParseBiosciences, cell fixation kit v3, CF100).Library Construction - Collected samples were processed for highly multiplexed single-cell RNA sequencing (scRNA-seq) using a split-pool combinatorial barcoding kit (ParseBiosciences, WT Mega kit v3, MG100).OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - 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. We used scanpy python package (v1.10.3).Sequence Alignment - We used Parse Biosciences Software (v1.3.1) to demultiplex barcodes, map to hg38 human transcriptome and generate count matrix.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq XRNA-seq of coding RNA from single cellsHomo sapiensNadezhda AzbukinaFátima Sanchís CallejafalsescRNA-seq of human neural brain organoids patterning reproducibility screeningIn order to systematically investigate, to which extent cell lines and experimental batches affect the consistency of organoid patterning, we conducted a multiplexed morphogen patterning experiment to study the reproducibility of the effects induced by retinoic acid, FGF-8, SHH and CHIR. We have tested influence of 2 neural induction methods, 2 experimental batches and 4 cell lines: H1 (XY), H9 (XX), WTC (XY) and WIBJ (XX) on cell type composition consistency after morphogen patterning using single-cell transcriptomic readout (Parse Bioscience) and pooling 3 organoids for each condition.2025-10-01T00:00:00Z2026-05-27T12:16:28.418Z2025-09-23T12:01:48.657ZE-MTAB-15622ERP180513EFO_0002944EFO_0004170EFO_0005684EFO_0004917EFO_0005518EFO_0003816EFO_0004184