7ff6de61-362b-496f-966b-a6d40d83be1e - samples
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ABSTRACT:
PROVIDER: EGAD00001005427 | EGA |
REPOSITORIES: EGA
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Project description:Adenine base editing (ABE) enable enzymatic conversion from A-T into G-C base pairs. ABE hold promise for clinical application, as it does not depend on the introduction of double-strand breaks, contrary to conventional CRISPR/Cas9-mediated genome engineering. Here we describe a cystic fibrosis (CF) intestinal organoid biobank, representing 646 patients, of which ~20% can theoretically be repaired by ABE. We apply the SpCas9-ABE (PAM recognition sequence: NGG) and the xCas9-ABE (PAM recognition sequence: NGN) on four selected CF organoid samples. Genetic and functional repair was obtained in all four cases, while whole genome sequencing (WGS) of corrected lines of two patients did not detect off-target mutations. These observations exemplify the value of large, patient-derived organoid biobanks representing hereditary disease, and indicate that ABE may be safely applied in human cells.
Whole genome sequencing of Cas9 repaired cystic fibrosis organoids
Three SpCas9-ABE (R785X/R785X) and three xCas9-ABE-repaired organoid clones (F508del/R553X) and their respective unrepaired control organoids were paired-end whole genome sequenced using Illumina Novaseq 6000 system. This WGS confirmed the Cas9 repair worked in these organoids and showed no off-target hits nor no mutational hotspots created by the repair system.
| EGAS00001003951 | EGA
Project description:Seven clonal organoid lines and one bulk wild-type control sample were paired-end whole-genome sequenced using the Illumina Novaseq 6000 system. We sequenced four clonal intestinal organoid lines harbouring engineered TP53 and FBXW7 mutations as well as three lines targeted for oncogenic APC/TP53/PIK3CA/SMAD4 mutations. The reads were mapped to hg38 genome assembly and data is provided as BAM files.
| EGAD00001009827 | EGA
Project description:Methods: Mouse bladder tumor organoids with Trp53, Pten, Rb1 mutation and Myc, KrasG12D amplification were treated with 10μM SRT1720 and Vehicle for 12 hours respectively. RNA libraries of SRT1720 and vehicle treated mouse bladder organoids were constructed according to standard Illumina protocols, and bulk RNA-seq were performed using Illumina NovaSeq 6000 sequencing platform.
2021-09-01 | GSE155525 | GEO
Project description:This study used snATAC-seq to profile Chromatin accessibility in 26 day-old iPSC-derived kidney organoids, treated with TGFB1, the EzH2 inhibitor GSK343, a combination of both or a vehicle control for 48 hours (days 24-26) before harvesting. 2 organoids per condition were pooled and dissociated using a cold-active protease. Nuclei were extracted and profiled using the 10X Genomics Single-cell ATAC reagent kit v1.1. Libraries were sequenced using paired-end reads on an Illumina NovaSeq 6000. Initial processing was performed using CellRanger ATAC v1.2.0 (10X Genomics).
2023-04-30 | E-MTAB-11139 | biostudies-arrayexpress
Project description:Purpose: The goal of this study is to compare NGS-derived transcriptomes of wild type mouse small interstinal organoids and organoids deficient for the enzyme acetyl-coa-carboxylase (ACC) 1 (Acaca). Methods: mRNA profiles were generated from organoids at 24h and 96h upon in vitro deletion of the Acaca gene and the respective non-deleted wild type controls in triplicates. Total RNA was isolated using RNeasy Micro Kit (Qiagen) and libraries were prepared using NEBNext Single Cell/Low Input RNA Library Prep (New England BioLabs). The libraries were sequenced on Illumina NovaSeq 6000 using NovaSeq 6000 S2 Reagent Kit (100 cycles, paired end run). Each FASTQ file gets a quality report generated by FASTQC tool. Before alignment to reference genome each sequence in the raw FASTQ files were trimmed on base call quality and sequencing adapter contamination using Trim Galore! wrapper tool. Reads shorter than 20 bp were removed from FASTQ file. Trimmed reads were aligned to the reference genome (GRCm38.) using open source short read aligner STAR (https://code.google.com/p/rna-star/) with settings according to log file. Results: the sequencing depth of our libraries is > 3 x107 reads per RNA sample. PCA analysis revealed a close relationship between WT and ACC1-deficient organoids at 24h, whereas samples at 96h were distinct from each other. Lack of ACC1 strongly reduced the expression of genes associated with intestinal epitelial stem cells. Moreover, GSEA revealed downregulation of pathways associated with DNA replication, cell cycle and chromosome segregation in ACC1-deficient organoids. Conclusions: Our study highlights the importance of ACC1-mediated cellular fatty acid synthesis for the maintenance of intestinal epithelial stem cells in mouse intestinal oganoids.
2021-11-10 | GSE188386 | GEO
Project description:Purpose: One goal of the bulk RNA-seq of organoids is to verify the differentially expressed genes between species identified by scRNA-seq .The other goal is to detected the differentially expressed genes when organoids from different species were treated with TNFα Methods: The total RNA from organoids was extracted by RNeasy Mini Kit (Qiagen). After total RNA extraction and then subjected to high-throughput sequencing on an Illumina Novaseq 6000 platform. Results: Most of the differentially expressed genes between species identified by bulk RNA-seq are consistent with the genes filtered by scRNA-seq. Conclusions: F3 and CLCA1 show higher expression in organoids of pig antral epithelium than in other three species. TNFα treatment upregulated the expression of immune response genes in all species
2023-09-11 | GSE225274 | GEO
Project description:We have developed a human small intestinal organoid (hSIO)-based mathematical modelling approach for the early prediction of GI toxicity of oral antiproliferative cancer treatments. Our mathematical model comprises hSIOs proliferative cells that become non-proliferative and eventually undergo cell death. Negative feedback loops regulate the rate of transition between these states, enhancing proliferation during the organoid expansion phase and balancing cell death in the stationary phase. Furthermore, the stationary phase is eventually followed by organoid decay, if organoids are not split and medium refreshment is not performed, which we modelled by accounting for the toxic effect of accumulated cell-produced waste material. Regarding drug-induced toxicity, the model implements cycle arrest and cell death of proliferative cells associated with exposure of oncology drugs
2026-05-01 | MODEL2509110001 | BioModels
Project description:Five edited and two unedited organoid clones with one clone prior to editing were paired-end whole genome sequenced using Illumina Novaseq 6000 system. The reads were mapped to hg38 genome assembly and data is provided as BAM files.
| EGAD00001007744 | EGA
Project description:Human nontransformed retinal pigment epithelia RPE-PPM1D-T2 cells carrying a truncating mutation in exon 6 of the PPM1D were exposed to ionising radiation (3 Gy) and subsequently were grown in semisolid media for 8 weeks. Six spheroid clones (RPE-PPM1D-T2-SA clones 1-6) were recovered and then were cultivated in adherent conditions. DNA was isolated from asynchronically growing parental RPE-PPM1D-T2 and transformed RPE-PPM1D-T2-SA-1 to 6 cells and was subjected to whole exome sequencing. DNA sequencing libraries were prepared using KAPA EvoPlus Kit (Roche) and were sequenced on the NovaSeq 6000 system using NovaSeq S1 Reagent Kit v1.5, 200 cycles (Illumina) with mean coverage >35 DNA samples, respectively. DNA fastq files were mapped to the hg19 reference using Novoalign (novoalign_2.08.03). PCR duplicates were removed from the BAM files using Picard Tools (picard-tools 1.129), and variant calling was performed using GATK HaplotypeCaller (3.8). Copy number variations (CNV) were analyzed using CNVkit version 0.7.4. Areas with median coverage >20 were included in the analysis. RNA fastq files were mapped to the hg19 reference using STAR (STAR-2.5.2b). The PCR duplicates were removed using Picard Tools (picard-tools 1.129).
2024-04-05 | E-MTAB-13923 | biostudies-arrayexpress
Project description:The data consists of 3 BAM files. Two of three BAMs are tumour FFPE samples (1 repaired-FFPE; 1 unrepaired-FFPE); The other BAM file is sequenced from normal colon tissue
| EGAD00001007716 | EGA