biostudies-arrayexpressFátima Sanchís CallejaHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15667This experiment seeks to unravel the effects of concentration, timing and combinatorial variables on the response to morphogens during early human neural organoid development. Human neural organoids derived from three different hESC lines were cultured individually and exposed to systematic variations of morphogen treatments. At the end of the 21-day culture period, organoids exposed to the same treatment were pooled (this defines \"one sample\") and dissociated into single cells. Each sample was then labelled with hashing antibodies to enable multiplexing while running the Chromium Next GEM Single Cell 3’ v3/3.1 kit (10X Genomics) workflow. Single-cell transcriptome libraries were then sequenced in SP/S1/S2 flowcells with Illumina NovaSeq technology, using paired-end 28/10/10/90 or 28/8/0/91 configuration (Read1/IDX i7/IDX i5/Read2).biostudies-arrayexpressSample Treatment - Morphogen treatments were applied from day 0 to day 20 of culture. Each condition featured a systematic variation in morphogen timing, concentration or combination.Nucleic Acid Extraction - Cells were lysed immediately after GEM generation as indicated in the 10X Genomics Chromium Next GEM Single Cell 3' v3.1 kit protocol.Sample Collection - Organoids were harvested after 21 days in culture and pooled prior to dissociation into single cells.Growth Protocol - hPSCs were aggregated into embryoid bodies in iPS Brew medium, then exposed to Neural Induction Medium for 14 days and finally changed into Neural Differentiation Medium with Vitamin A until day 21.Sequencing - 10x libraries were sequenced in SP/S1/S2 flowcells with Illumina NovaSeq technology, using paired-end 28/10/10/90 or 28/8/0/91 configuration (Read1/IDX i7/IDX i5/Read2).Library Construction - Once cells were encapsulated in GEMs, an RT step was run to convert polyadenylated mRNAs into full-length cDNA sequences with a UMI and cellular barcode. cDNA products were then purified with magnetic beads (Dynabeads MyOne SILANE, manufactured by Thermo Fisher but included in 10X Genomics kits) and further amplified in an extra PCR step using a partial TruSeq read 1 and partial TSO (Template-Switch Oligo) as primers. Another purification step based on construct size was performed with SPRIselect reagent (Beckman Coulter) prior to quantification and quality control with a Bioanalyzer High Sensitivity chip (2100 Bioanalyzer, Agilent). One-quarter of the cDNA from each 10X sample was carried over for gene expression library construction. In short, cDNA was fragmented to eliminate the TSO incorporated in the previous step and ends were repaired and A-tailed. Reaction products were purified with SPRIselect reagent and Truseq read 2 adaptors were ligated, one of them being a partial read to provide an overhang for the following PCR. Another cleanup step with SPRIselect was performed before the Sample Index PCR when P5 and P7 sequences (for Illumina sequencing) and sample indices (to allow for multiplexing in the sequencer) were included in the construct. The final product was purified with SPRIselect magnetic beads and a concentration and quality check were performed by running a Bioanalyzer High Sensitivity chip before sequencing. OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesSequence Alignment - After sample demultiplexing, cell ranger v3.1.0 was used with GRCh38-3.0.0 as a reference to derive gene-cell count matrices from the sequencing read (fastq) files of the gene expression library.Data Transformation - The function SCTransform from the package Seurat was used to normalize and scale gene count matrices.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000RNA-seq of coding RNA from single cellsHomo sapiensSystematic scRNA-seq screens profile neural organoid response to morphogensBarbara TreutleinFátima Sanchís-Calleja, Nadezhda Azbukina, Akanksha Jain, Zhisong He, Ryoko Okamoto, Charlotte Rusimbi, Pedro Rifes, Gaurav Singh Rathore, Malgorzata Santel, Jasper Janssens, Makiko Seimiya, Jonas Fleck, Agnete Kirkeby, J. Gray Camp, Barbara TreutleinFátima Sanchís CallejaNadezhda AzbukinafalseSingle-cell RNA sequencing of developing human neural organoids exposed to an array of morphogen treatments varying morphogen concentration, timing and combinatoricsThis experiment seeks to unravel the effects of concentration, timing and combinatorial variables on the response to morphogens during early human neural organoid development. Human neural organoids derived from three different hESC lines were cultured individually and exposed to systematic variations of morphogen treatments. At the end of the 21-day culture period, organoids exposed to the same treatment were pooled (this defines \"one sample\") and dissociated into single cells. Each sample was then labelled with hashing antibodies to enable multiplexing while running the Chromium Next GEM Single Cell 3’ v3/3.1 kit (10X Genomics) workflow. Single-cell transcriptome libraries were then sequenced in SP/S1/S2 flowcells with Illumina NovaSeq technology, using paired-end 28/10/10/90 or 28/8/0/91 configuration (Read1/IDX i7/IDX i5/Read2).2025-10-15T00:00:00Z2026-04-13T10:05:43.742Z2025-10-13T11:33:46.919ZE-MTAB-15667ERP182047E-MTAB-15622EFO_0002944EFO_0004170EFO_0003789EFO_0005684EFO_0004917EFO_0005518EFO_0003816EFO_0004184EFO_0003969