{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Rajib Schubert"],"organism":["Homo sapiens"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-16805"],"description":["This study benchmarks bulk and single-cell long-read RNA sequencing technologies in a human neuronal model of Fragile X syndrome. NGN2-induced neurons were generated from patient-derived iPSCs carrying a silenced FMR1 gene (FXS line E3) and an isogenic CRISPR-corrected rescue line (IsoB11) in which FMR1 expression is restored. These conditions provide a defined system to evaluate transcript detection and quantification across sequencing platforms.   Bulk and single-cell RNA-seq datasets were generated using Illumina short-read sequencing and long-read sequencing from Pacific Biosciences (PB) and Oxford Nanopore Technologies (ONT). Single-cell libraries were prepared using the 10x Genomics Chromium platform. ERCC and SIRV spike-in controls were added to bulk samples to enable benchmarking of transcript quantification accuracy.   Three biological replicates were sequenced for each condition. The dataset enables cross-platform comparisons of transcript detection, quantification methods, transcript length biases, and sequencing depth requirements for long-read transcriptomic analyses."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Sample Collection - NGN2-induced glutamatergic neurons were generated from Fragile X syndrome patient-derived induced pluripotent stem cells (iPSCs) and an isogenic CRISPR-corrected rescue line. Neuronal differentiation was performed for 21 days using doxycycline-inducible NGN2 expression. Cells were cultured on polyornithine/laminin coated plates and maintained with BDNF-containing neuronal medium. Cells were harvested at day 21 for downstream single-cell and bulk RNA sequencing.","Sequencing - Illumina single-cell libraries were sequenced on an Illumina NovaSeq 6000 platform using a standard 10x Genomics configuration.","Sequencing - PacBio single-cell libraries were sequenced on a PacBio Revio platform using the Revio Polymerase Kit (102-817-600) with 24-hour movie times to generate high-fidelity (HiFi) reads.","Nucleic Acid Extraction - For single-cell RNA-seq, cell viability was confirmed to exceed 95% prior to processing. Approximately 6,000 cells per replicate were targeted using the 10x Genomics Chromium Next GEM Single Cell 3' Kit v3.1. Following Post-GEM-RT Cleanup and cDNA amplification, the resulting cDNA was utilized for all downstream short- and long-read library preparations.","Library Construction - 10 ng of single-cell cDNA generated from the 10x Genomics Chromium NextGEM Single-Cell 3′ Kit v3.1 workflow was used as input. Amplified cDNA was processed according to the Oxford Nanopore single-cell transcriptomics protocol (SST_v9198_v114_revE), including pull-down of biotin-tagged cDNA using M280 magnetic streptavidin-coated beads and a second PCR round using PRM primers. Following QC, libraries were prepared using the Ligation Sequencing Kit V14 (SQK-LSK114).","Library Construction - Amplified single-cell cDNA (75 ng) from the 10x Genomics Chromium NextGEM Single-Cell 3′ Kit v3.1 workflow was used for PacBio library preparation across two sequencing rounds. Libraries were generated using the MAS-Seq for 10x 3’ Concatenation Kit (PB, 102-407-900) or the Kinnex Single-Cell RNA Kit (PB, 103-072-200) according to the manufacturer’s instructions. SMRTbell libraries were purified with SMRTbell Cleanup Beads (PB, 102-158-300).","Sequencing - ONT single-cell libraries were sequenced on PromethION flow cells (FLO-PRO114M). Basecalling was executed with the dorado dna_r10.4.1_e8.2_400bps_hac@v4.3.0 model.","Library Construction - Single-cell libraries were generated using the 10x Genomics Chromium platform with the NextGEM Single Cell 3′ v3.1 kit following the manufacturer’s protocol. Approximately 6,000 cells were loaded per channel to generate barcoded single-cell cDNA. Following reverse transcription and cDNA amplification, libraries were prepared for Illumina sequencing via enzymatic fragmentation, end repair, A-tailing, and adapter ligation. Unique i5 and i7 indices were incorporated using the Dual Index Kit TT Set A. Libraries were validated using an Agilent TapeStation D1000."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","Processed Data","MAGE-TAB Files"],"data_protocol":["Data Transformation - PacBio reads were aligned to the human transcriptome (hg38, GENCODE V45) and quantified using oarfish.","Data Transformation - PacBio uBAM tags were extracted using pysam and saved as compressed TSV files, to enable retagging of uBAMs after download from common repositories.","Data Transformation - Illumina reads were quantified against GENCODE V45 using simpleaf.","Data Transformation - ONT reads were aligned to the human transcriptome (hg38, GENCODE V45) and quantified using oarfish."],"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["Illumina NovaSeq 6000","Sequel IIe","PromethION"],"study_type":["RNA-seq of coding RNA from single cells"],"species":["Homo sapiens"],"pubmed_authors":["Rajib Schubert"],"additional_accession":[]},"is_claimable":false,"name":"Single cell short- and long-read RNA-sequencing of iNeurons in Fragile-X and control cell-lines","description":"This study benchmarks bulk and single-cell long-read RNA sequencing technologies in a human neuronal model of Fragile X syndrome. NGN2-induced neurons were generated from patient-derived iPSCs carrying a silenced FMR1 gene (FXS line E3) and an isogenic CRISPR-corrected rescue line (IsoB11) in which FMR1 expression is restored. These conditions provide a defined system to evaluate transcript detection and quantification across sequencing platforms.   Bulk and single-cell RNA-seq datasets were generated using Illumina short-read sequencing and long-read sequencing from Pacific Biosciences (PB) and Oxford Nanopore Technologies (ONT). Single-cell libraries were prepared using the 10x Genomics Chromium platform. ERCC and SIRV spike-in controls were added to bulk samples to enable benchmarking of transcript quantification accuracy.   Three biological replicates were sequenced for each condition. The dataset enables cross-platform comparisons of transcript detection, quantification methods, transcript length biases, and sequencing depth requirements for long-read transcriptomic analyses.","dates":{"release":"2025-03-30T00:00:00Z","modification":"2026-04-25T06:05:03.874Z","creation":"2026-03-27T17:44:47.894Z"},"accession":"E-MTAB-16805","cross_references":{"ENA":["ERP191491"],"EFO":["EFO_0002944","EFO_0004170","EFO_0005684","EFO_0005518","EFO_0003816","EFO_0004184"]}}