biostudies-arrayexpressErick Andrés Muciño OlmosHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15002Neuroblastoma (NB) is a pediatric cancer characterized by significant heterogeneity and poor prognosis, particularly in high-risk cases with MYCN amplification. Understanding the molecular response of neuroblastoma to different treatments can provide valuable insights into potential therapeutic strategies. This experiment assessed the in vivo response to treatment with BAY390 and A967079. A neuroblastoma in vivo model was established via subcutaneous injection of human high-risk, MYCN-amplified neuroblastoma organoids into female NSG mice (PDX, LU-NB-3R). Once tumors reached 150 mm³, mice were randomized into treatment groups: control (n=8), A967079 (n=6), and BAY390 (n=7). All drugs were administered via oral gavage at the following concentrations: A967079 (300 mg/kg, once daily) or BAY390 (90 mg/kg, once daily), with a vehicle control group. Treatment was administered for 21 days, after which mice were euthanized. Tumor samples were collected in RNA later and frozen at -80 °C until RNA extraction. RNA was extracted from tumor tissue, and RNA sequencing was performed.biostudies-arrayexpressNucleic Acid Extraction - Total RNA was extracted using the Tissuelyzer (85600, QIAGEN) and the AllPrep DNA/RNA Mini kit (80204, QIAGEN) according to the manufacturer’s instructions. Quality was assessd by using the Agilent RNA ScreenTape Assay (G2991-90020 Rev.B) on the TapeStation 4200 System (Agilent). Concentration was measured with the QuantIT RNA High Sensitivity Assay Kit (Q33140, Thermo Fisher Scientific) using the Qubit Flex Fluorometer (Q33327, Invitrogen, Thermo Fisher Scientific).Library Construction - Library preparation was performed according to the Illumina Stranded mRNA pep Ligation reference Guide (Document # 1000000124518 v03) with the change to 12 PCR cycles at the DNA fragment enrichment. Reagents used were the Illumina Standed MRNA Prep, Ligation (2004053, Illumina). Equipment used was the KingFisher FLEX (18-5400620, ThermoFisher) for automatized cleanup steps. Incubations and PCR were performed on the Eppendorf Mastercycler X50s (6311000010, Eppendorf). Library quality was measured according to the Agilent D5000 ScreenTape System Quick Guide, Part Number: G2964-90050 Rev. B, Edition 09/2015 using the Tape Station 4200 (Agilent) and D5000 ScreenTape (5067- 5588) with D5000 Reagents (5067- 558, Agilent). The concentration was measured using the Qubit Flex (Q33327, Invitrogen, Thermo Fisher Scientific) with the QuantIT 1X dsDNA HS Assay Kit (Q33232, Thermo Fisher Scientific) according to the Qubit Flex Fluorometer Quick Reference No. MAN0018187 Rev. A.0 protocol.Sample Treatment - Neuroblastoma in vivo model of LU-NB-3R was obtained via subcutaneous injection of human high-risk, MYCN-amplified neuroblastoma organoids in female NSG mice. When tumors reached 150 mm3, mice were randomized into treatment groups: control (n=8), A967079 (n=6), and BAY390 (n=7). All drugs were administered via oral gavage at the following concentrations: A967079 (300 mg/kg, once daily), BAY 390 (90 mg/kg, once daily) or vehicle for the control group. Treatment was administered for 21 days, after which mice were euthanized. All mice tolerated the treatment.Sample Collection - Treatment was administered for 21 days, after which mice were euthanized, tumor pieces were resected, cut to 3x3 mm pieces on ice and placed in RNA later-ICE transition solution (AM7030, Invitrogen) overnight.Sequencing - Library input to sequencing: 0.65 nM PhiX Control input (FC-110-3001, Illumina): 1% Sequencing setup (read one - index reads - read two, bp): 150-10-10-150 Protocol: NovaSeq 6000 Sequencing System Guide (Document # 1000000019358 v11) Reagent/kit: NovaSeq 6000 S4 Reagent Kit, 300 cycles v1.5 (20028312, Illumina) Equipment: NovaSeq 6000 System (20012850, Illumina)OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData 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 (v113) transcriptome via the biomaRt R package (v2.58.0; Durinck et al., 2009). To correct for unwanted sources of variation, we applied RUVs (Remove Unwanted Variation using Replicates) (Risso et al., 2014). This method estimates factors of unwanted variation using replicate samples where the covariates of interest remain constant. First, a SeqExpressionSet was constructed from the raw count matrix after filtering out lowly expressed genes that were not present in at least the minimum number of samples for a group. Upper-quartile normalization was then applied using the betweenLaneNormalization function. A design matrix (~ condition) was specified, and three factors of unwanted variation (k=3) were estimated using the RUVs function, considering all genes to improve robustness. The corrected gene-level count matrix was then imported into a DESeqDataSet object and normalized using the DESeq2 package (v1.42.0; Love et al., 2014). DESeq2 normalization was applied after accounting for the estimated factors of unwanted variation, using the design formula ~ W_1 + W_2 + W_3 + condition. For differential expression analysis, the DESeq2 workflow was followed, and log fold changes were moderated using the lfcShrink() function with type = \"apeglm\" (Zhu et al., 2019) to improve effect size estimation and reduce variability in low-expression genes.Sequence Alignment - After trimming, sequencing reads were pseudoaligned using the Kallisto tool (v0.51.1; Bray et al., 2016). The pseudoalignment was performed against the GRCh38 (v113) 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.UnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000RNA-seq of coding RNAHomo sapiensErick Andrés Muciño OlmosfalseRNA-Seq analysis of subcutaneous patient-derived neuroblastoma xenografts (LU-NB-3R): Comparison of BAY390, A967079, and vehicle controlsNeuroblastoma (NB) is a pediatric cancer characterized by significant heterogeneity and poor prognosis, particularly in high-risk cases with MYCN amplification. Understanding the molecular response of neuroblastoma to different treatments can provide valuable insights into potential therapeutic strategies. This experiment assessed the in vivo response to treatment with BAY390 and A967079. A neuroblastoma in vivo model was established via subcutaneous injection of human high-risk, MYCN-amplified neuroblastoma organoids into female NSG mice (PDX, LU-NB-3R). Once tumors reached 150 mm³, mice were randomized into treatment groups: control (n=8), A967079 (n=6), and BAY390 (n=7). All drugs were administered via oral gavage at the following concentrations: A967079 (300 mg/kg, once daily) or BAY390 (90 mg/kg, once daily), with a vehicle control group. Treatment was administered for 21 days, after which mice were euthanized. Tumor samples were collected in RNA later and frozen at -80 °C until RNA extraction. RNA was extracted from tumor tissue, and RNA sequencing was performed.2026-04-01T00:00:00Z2026-04-02T01:04:58.594Z2025-04-04T09:29:38.118ZE-MTAB-15002ERP171263EFO_0002944EFO_0004170EFO_0004917EFO_0005518EFO_0003816EFO_0003738EFO_0004184EFO_0003969