{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Anne Grapin-Botton"],"organism":["Homo sapiens"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-14625"],"description":["H9 human embryonic stem cells with one allele of the PDX1 gene genetically modified into a reporter PDX1-P2A-H2B-GFP and one allele of NEUROG3 gene genetically modified into NEUROG3-P2A-tagRFP-T-NLS were differentiated into the pancreatic lineage until Stage 4 Day 3 of the in vitro differentiation protocol (Rezania et al., 2014, as modified in Petersen et al., 2017) and expanded as pancreatic progenitor (PP) organoids as described in Gonçalves et al 2021. PP organoids were seeded on day 0 and treated with 3 µM CHIR 99021 in the expansion medium from day 3 to day 10, with DMSO as negative control. Organoids from treated and control conditions were harvested on day 4 and day 10 for gene expression analysis by bulk RNA sequencing. d10_CHIR_7days samples were treated with CHIR from day 3 to day 10, whereas d10_CHIR_4days samples were treated with CHIR from day 3 to day 7, followed by DMSO from day 7 to day 10."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Sequencing - Sequencing was performed after quantification with Fragment Analyzer, on Illumina Novaseq 6000 in 100 bp paired-end mode to a depth 30 million fragments per library.","Nucleic Acid Extraction - Total RNA was subjected to a modified version of SMARTseq-2 protocol published by Picelli et al. 2013 (doi: 10.1038/nmeth.2639). In short, total RNA was isolated with RNeasy Micro kit (Qiagen) and eluted in nuclease free water.","Library Construction - mRNA was isolated from on average 450 ng total RNA by poly-dT enrichment using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB) according to the manufacturer’s instructions. Samples were then directly subjected to the workflow for strand-specific RNA-Seq library preparation (Ultra II Directional RNA Library Prep, NEB). For ligation NEB Next Adapter for Illumina of the NEB Next Multiplex Oligos for Illumina Kit were used. After ligation, adapters were depleted by an XP bead purification (Beckman Coulter) adding the beads solution in a ratio of 0.9:1 to the samples. Unique dual indexing was done during the following PCR enrichment (12 cycles) using amplification primers carrying the same sequence for i7 and i5 index (i5: AAT GAT ACG GCG ACC ACC GAG ATC TAC AC NNNNNNNN ACA TCT TTC CCT ACA CGA CGC TCT TCC GAT CT, i7: CAA GCA GAA GAC GGC ATA CGA GAT NNNNNNNN GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC T). After two more XP bead purifications (0.9:1), libraries were quantified using the Fragment Analyzer (Agilent).","Sample Collection - hESC-derived PP-organoids (Gonçalves et al 2021) were dissociated into single cells with TrypLE and snap-frozen for RNA extraction."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","Processed Data","MAGE-TAB Files"],"data_protocol":["Sequence Alignment - Paired-end (2x101) Illumina read data sets were processed. Illumina universal adapters were trimmed from both ends of the reads with Cutadapt v1.16 discarding reads shorter trimmed to a length shorter than 19 nt. Quality of reads was assessed using FastQC 0.11.9. Reads were mapped against the Homo sapiens genome reference assembly GRCh38 and genes of the Ensembl release v99 were quantified using STAR 2.7.3a (settings: --outSAMstrandField intronMotif  --outFilterMultimapNmax 1 --outSJfilterCountUniqueMin 3 1 1 1 --quantMode GeneCounts --alignSJDBoverhangMin 5 --alignSJoverhangMin 8 --outFilterMultimapNmax 1 --outFilterMismatchNmax 999 --outFilterMismatchNoverLmax 0.1 --alignMatesGapMax 200000 --seedSearchStartLmax 25 --chimSegmentMin 20 --twopassMode Basic --alignIntronMin 20 --alignIntronMax 200000).","Data Transformation - A genes-by-samples matrix (gene_counts.txt) with read counts was constructed by concatenating the read counts per sample the STAR 2.7.3a mapper dir output running it with argument --quantMode GeneCounts. For more details see the alignment protocol."],"omics_type":["Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["Illumina NovaSeq 6000"],"study_type":["RNA-seq of coding RNA"],"species":["Homo sapiens"],"pubmed_authors":["Anne Grapin-Botton","Rashmiparvathi Keshara"],"additional_accession":[]},"is_claimable":false,"name":"Bulk RNA sequencing of pancreatic progenitor spheroids treated with CHIR99021 and control (DMSO) conditions","description":"H9 human embryonic stem cells with one allele of the PDX1 gene genetically modified into a reporter PDX1-P2A-H2B-GFP and one allele of NEUROG3 gene genetically modified into NEUROG3-P2A-tagRFP-T-NLS were differentiated into the pancreatic lineage until Stage 4 Day 3 of the in vitro differentiation protocol (Rezania et al., 2014, as modified in Petersen et al., 2017) and expanded as pancreatic progenitor (PP) organoids as described in Gonçalves et al 2021. PP organoids were seeded on day 0 and treated with 3 µM CHIR 99021 in the expansion medium from day 3 to day 10, with DMSO as negative control. Organoids from treated and control conditions were harvested on day 4 and day 10 for gene expression analysis by bulk RNA sequencing. d10_CHIR_7days samples were treated with CHIR from day 3 to day 10, whereas d10_CHIR_4days samples were treated with CHIR from day 3 to day 7, followed by DMSO from day 7 to day 10.","dates":{"release":"2025-11-14T00:00:00Z","modification":"2025-11-14T02:01:59.42Z","creation":"2024-11-21T23:36:45.794Z"},"accession":"E-MTAB-14625","cross_references":{"ENA":["ERP166426"],"EFO":["EFO_0002944","EFO_0004170","EFO_0004917","EFO_0005518","EFO_0003816","EFO_0003738","EFO_0004184"]}}