{"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-16791"],"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.","Nucleic Acid Extraction - Total RNA was extracted from NGN2-induced neurons using TRIzol reagent according to the manufacturer’s instructions. RNA concentration was measured using Qubit RNA High Sensitivity assays and RNA integrity was assessed using Agilent ScreenTape analysis. Samples used for sequencing had RNA integrity numbers (RIN) greater than 9.","Sequencing - Libraries were sequenced on a PromethION 24 instrument using PromethION flow cells (FLO-PRO114M). Samples were multiplexed with three replicates per flow cell. Basecalling was performed using the Dorado dna_r10.4.1_e8.2_400bps_hac@v4.3.0 model.","Library Construction - For each replicate, 250 ng of total RNA was used as input. Libraries were prepared using the TruSeq Stranded mRNA library preparation kit (Illumina, 20020595) to capture polyadenylated transcripts while maintaining strand specificity.","Sequencing - Sequencing was conducted on a PacBio Revio platform using the Revio Polymerase Kit (102-817-600). Libraries were loaded via diffusion and sequenced with a 24-hour movie time per SMRT Cell. Replicates were multiplexed at a rate of two per SMRT Cell.","Library Construction - Library preparation was performed using 300 ng of total RNA (RIN > 7) as input. High-quality cDNA was generated using the Iso-Seq Express 2.0 Kit (PB, 103-071-500) and further processed with the Kinnex PCR 8-fold Kit (PB, 103-071-600) for multiplexing. Following ten cycles of cDNA amplification, libraries were purified using SMRTbell Cleanup Beads (PB, 102-158-300). Final library concentration and fragment size distribution were validated via Agilent TapeStation 4200 and Qubit Flex Fluorometer.","Sequencing - Libraries were sequenced on an Illumina NovaSeq X Plus platform. The system was configured for paired-end 150 bp (PE150) reads, aiming for a sequencing depth of 50 million reads per sample.","Library Construction - ng of total RNA, inclusive of spike-in controls, was prepared using the cDNA-PCR Sequencing V14 kit (SQK-PCS114.24). The protocol included 14 cycles of PCR amplification to generate sufficient material for nanopore sequencing."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","Processed Data","MAGE-TAB Files"],"data_protocol":["Data Transformation - Illumina reads were quantified using Salmon and optionally corrected for inferential relative variance using edgeR.","Data Transformation - PacBio reads were aligned to the human genome (hg38) and quantified using Isosceles.","Data Transformation - ONT reads were aligned to the human genome (hg38) and quantified using Isosceles.","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."],"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["Illumina NovaSeq X","Sequel IIe","PromethION"],"study_type":["RNA-seq of coding RNA"],"species":["Homo sapiens"],"pubmed_authors":["Rajib Schubert"],"additional_accession":[]},"is_claimable":false,"name":"Bulk 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":"2026-03-25T00:00:00Z","modification":"2026-04-24T18:27:16.686Z","creation":"2026-03-20T21:39:01.774Z"},"accession":"E-MTAB-16791","cross_references":{"ENA":["ERP191112"],"EFO":["EFO_0002944","EFO_0004170","EFO_0005518","EFO_0003816","EFO_0003738","EFO_0004184"]}}