biostudies-arrayexpressElisa BalmasHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15308iPS2-10X-seq on Neural organoid differentiation after 35days of differentiation. This experiment is a proof of principle that iPS2seq can be applied to different differentiation strategies without risk of silencing. This experiment also showed how neuro-iPSC clones differentiate preferentially towards specific neuronal lineages, which are essential to detect and exclude from the analysis to avoid misleading results.biostudies-arrayexpressLibrary Construction - CG000388 Rev B (with cell multiplexing oligos). hPS-2seq barcode enrichment protocol was performed with custom primers binding on the TET repressor.Sample Collection - Human neural organoids were cultured for 30 days following the protocol outlined by Velasco et al. (2019) PMID: 31168097. iPS2seq transfection pools were differentiated separately, and tetracycline was added to the treated organoids throughout the culture. Organoids were dissociated into single-cell suspensions using papain-based enzymatic digestion as described in Protocol Exchange (https://protocolexchange.researchsquare.com/article/pex-258/v1). Cell counts and live cell estimation were determined using a hemocytometer, and suspensions were adjusted to 2 million cells per tube. Then cells were labeled with CMO, sorted for vital cells and then loaded on the 10X controller.Nucleic Acid Extraction - Cell Multiplexing Oligo (CMO) labeling was performed according to the manufacturer’s protocol (CG000391) with an additional washing step. Cells were pooled in equal quantities, and the cell-pooled suspension was counted to ensure a final concentration of 1,600 cells/μL to obtain a final cell recovery of 30000 cells. Single-cell libraries were prepared using the Chromium Next GEM Single Cell 3' v3.1 protocol (CG000388 Rev C). Cells were loaded on a chip G.Sequencing - Nextseq 1000 P2 100 cycles with cycle allocation suggested by 10X in the user guide. Cell multiplexing barcodes were pulled in molar ratio compared to the gen expression library 1:6. iPS2seq barcodes were pulled in molar ratio compared to the gen expression library 1:20. Sequencing as 650pM loading concentration on an Illumina P2 xleap NextSeq 1000 platform using the recommended cycle configuration (R1: 28 cycles; i7: 10 cycles; i5: 10 cycles; R2: 90 cycles) with a 1% PhiX spike-in.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - shRNA information is atached in rc_barcodes_genes.csv to allow running CatcheR.Data Transformation - Data provided are row. Use the atached aggregation.csv and config.csv files to run cell ranger aggr (aggregation.csv) and then cell ranger multi (config.csv)Data Transformation - an RData file is atached with the full processed data. Data can be read in R and then it can be read and used with monocle 3. Additional txt files with data annotation and count data are provided to be analyzed with different platforms.UnknownTranscriptomicsGenomicsProteomicsNextSeq 1000RNA-seq of coding RNA from single cellsHomo sapiensElisa BalmasfalseiPS2-10X-seq Neural organoid differentiationiPS2-10X-seq on Neural organoid differentiation after 35days of differentiation. This experiment is a proof of principle that iPS2seq can be applied to different differentiation strategies without risk of silencing. This experiment also showed how neuro-iPSC clones differentiate preferentially towards specific neuronal lineages, which are essential to detect and exclude from the analysis to avoid misleading results.2025-10-27T00:00:00Z2025-10-27T08:43:10.28Z2025-07-03T16:16:36.517ZE-MTAB-15308ERP174607E-MTAB-15307EFO_0002944EFO_0004170EFO_0005684EFO_0005518EFO_0003816EFO_0004184