{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"submitter":["Erick Muciño Olmos"],"instrument_platform":["Illumina NovaSeq 6000"],"study_type":["RNA-seq of total RNA"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-14846"],"description":["Neuroblastoma (NB) is a pediatric cancer characterized by significant heterogeneity and poor prognosis, especially in high-risk cases with MYCN amplification. Understanding the molecular response of neuroblastoma to different treatments can provide insights into potential therapeutic strategies. The aim of the experiment was to evaluate the effects of treatment of pitavastatin (PIT), prochloperazine (PCZ) and combination (PIT + PCZ) on LU-NB-2 PDX derived organoid model. In our experiments we observed synergistic effects on cell viability and cell death of those two compounds and further RNA-seq was intended to decipher effects of the combination on the neuroblastoma cells. Experimental Workflow:  Sample Preparation: Tumor organoids were generated from PDX models of high-risk neuroblastoma patients with MYCN amplification.   Treatment: LU-NB-2 PDX organoids were dissociated to single cells aseeded in T25 flasks, allowed to grow for 24 h, and treated for 48 h with the combination of PCZ +PIT (1.6 µM and 6.7 µM, n = 8), PCZ only (1.6 µM, n = 8), PIT only (6.7 µM, n = 8), or DMSO (n = 8). The treatment resulted in a ~15% decrease in NB cell viability in the combination group.  RNA Extraction: Total RNA was extracted from the treated and control organoids.  Library Preparation and Sequencing: RNA-seq libraries were prepared and sequenced to generate high-throughput sequencing data.  Data Analysis: Differential gene expression analysis was performed to compare the treated samples against the untreated controls, aiming to elucidate the molecular effects of each treatment."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Growth Protocol - Cells were cultured and passaged as described (Persson et al., PMID: 28860499). LU-NB-2 PDX cells were seeded in T25 flasks (1milion cells per condition) and allowed to grow for 24 hours.","Sample Collection - Following the treatment period, cell pellets were collected by centrifugation. The samples were flash-frozen and stored at -80°C to preserve RNA integrity until further processing.","Sample Treatment - Following the initial growth period, LU-NB-2 PDX cells were treated for 48 hours with one of the following conditions: a combination of PCZ+PIT (1.6 µM + 6.7 µM, n = 4), PCZ alone (1.6 µM; n = 4), PIT alone (6.7 µM; n = 4), or DMSO as a vehicle control (1% v/v, n = 4). Two biological replicates were performed resulting in a total of 8 samples per each condition. Notably, treatment with the combination of PCZ and PIT led to an approximately 15% reduction in neuroblastoma cell viability compared to controls.","Nucleic Acid Extraction - Total RNA was extracted from the collected cell pellets using the AllPrep DNA/RNA Mini Kit (80204, Qiagen), following the manufacturer’s instructions. RNA quality and concentration were assessed before library preparation. RNA quality was evaluated using the Agilent RNA ScreenTape Assay (G2991-90020 Rev. B) on the TapeStation 4200 System (Agilent), while RNA concentration was measured with the QuantIT RNA HS Assay Kit (Q33140, Life Technologies) using the Qubit Flex Fluorometer (Q33327, Invitrogen Thermo Fisher Scientific).","Library Construction - Approximately 500 ng of total RNA was used for mRNA library preparation. Libraries were constructed using the TruSeq Stranded mRNA Library Prep Kit (20020594, Illumina) following the TruSeq Stranded mRNA Sample Preparation Guide (Part #15031047 Rev. E). The libraries were indexed with IDT for Illumina TruSeq RNA UD Indexes (20022371, Illumina) and subjected to 13 PCR cycles. Cleanup steps were automated on a KingFisher FLEX system (Thermo Scientific), and incubations and PCR were performed using an Eppendorf Mastercycler X50s (Eppendorf). Library concentrations were measured using the QuantIT 1X dsDNA HS Assay Kit (Q33232, Invitrogen) on a Qubit Flex Fluorometer (Q33327, Invitrogen Thermo Fisher Scientific). The Agilent D5000 ScreenTape System (G2964-90050 Rev. B) assessed library size and quality on the TapeStation 4200 System (Agilent).","Sequencing - Prepared RNA libraries were pooled and diluted to a final concentration of 0.65 nM, with 1% PhiX Control in the pool. Sequencing was performed on a NovaSeq 6000 System (Illumina) at the Center for Translational Genomics, Lund University. Libraries were sequenced using the NovaSeq 6000 S1 Reagent Kit, 300 cycles v1.5 (20028317, Illumina), following the NovaSeq 6000 Sequencing System Guide (Document #1000000019358 v11). Paired-end reads were generated with a configuration of 150-8-8-150 bp."],"figure_sub":["MIAME Score","Organization","Assays and Data","MAGE-TAB Files"],"pubmed_authors":["Erick Muciño Olmos"],"data_protocol":["Data Transformation - Transcript-level abundance estimates (TPM) were obtained from Kallisto during pseudoalignment. For downstream analyses, the pseudoalignment output files were summarized into gene-level counts using the tximport R package (v1.30.0; Soneson et al., 2015), with annotations derived from the Ensembl GRCh38 (v109) transcriptome via the biomaRt R package (v2.58.0; Durinck et al., 2009). The gene-level count matrix was subsequently imported into a DESeqDataSet object and normalized using the DESeq2 package (v1.42.0; Love et al., 2014), which applies a median-of-ratios method to account for differences in sequencing depth and RNA composition. The regularized log transformation (rlog) was applied for exploratory data visualization, such as principal component analysis (PCA). However, differential expression analysis was performed using the DESeq2 normalization protocol as per the recommended workflow in the DESeq2 vignette.","Sequence Alignment - After trimming, sequencing reads were pseudoaligned using the Kallisto tool (v0.48.0; Bray et al., 2016). The pseudoalignment was performed against the GRCh38 (v109) Homo sapiens transcriptome obtained from the Ensembl database (Cunningham et al., 2022). The transcriptome index was generated using both the cDNA and ncRNA FASTA files to ensure comprehensive coverage of transcriptomic features."],"additional_accession":[]},"is_claimable":false,"name":"RNA-seq of tumor organoids from patient-derived xenograft (PDX, LU-NB-2) of high-risk, MYCN-amplified neuroblastoma treated with prochlorperazine (PCZ), pitavastatin (PIT), the combination of both drugs, and untreated controls.","description":"Neuroblastoma (NB) is a pediatric cancer characterized by significant heterogeneity and poor prognosis, especially in high-risk cases with MYCN amplification. Understanding the molecular response of neuroblastoma to different treatments can provide insights into potential therapeutic strategies. The aim of the experiment was to evaluate the effects of treatment of pitavastatin (PIT), prochloperazine (PCZ) and combination (PIT + PCZ) on LU-NB-2 PDX derived organoid model. In our experiments we observed synergistic effects on cell viability and cell death of those two compounds and further RNA-seq was intended to decipher effects of the combination on the neuroblastoma cells. Experimental Workflow:  Sample Preparation: Tumor organoids were generated from PDX models of high-risk neuroblastoma patients with MYCN amplification.   Treatment: LU-NB-2 PDX organoids were dissociated to single cells aseeded in T25 flasks, allowed to grow for 24 h, and treated for 48 h with the combination of PCZ +PIT (1.6 µM and 6.7 µM, n = 8), PCZ only (1.6 µM, n = 8), PIT only (6.7 µM, n = 8), or DMSO (n = 8). The treatment resulted in a ~15% decrease in NB cell viability in the combination group.  RNA Extraction: Total RNA was extracted from the treated and control organoids.  Library Preparation and Sequencing: RNA-seq libraries were prepared and sequenced to generate high-throughput sequencing data.  Data Analysis: Differential gene expression analysis was performed to compare the treated samples against the untreated controls, aiming to elucidate the molecular effects of each treatment.","dates":{"release":"2025-05-25T00:00:00Z","modification":"2025-09-10T00:01:57.802Z","creation":"2025-02-14T16:36:58.996Z"},"accession":"E-MTAB-14846","cross_references":{"EFO":["EFO_0002944","EFO_0004170","EFO_0009653","EFO_0003789","EFO_0004917","EFO_0005518","EFO_0003816","EFO_0004184","EFO_0003969"]}}