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| Status |
Public on Dec 01, 2023 |
| Title |
RNA_AKP_Day5_STAR-pos-to-pos_3 |
| Sample type |
SRA |
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| Source name |
AKP organoid
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| Organism |
Homo sapiens |
| Characteristics |
tissue: AKP organoid
cell type: Colorectal cancer
time: Day 5
star day 0: pos
star day 5/12: pos
technical replicate: 3
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| Treatment protocol |
Organoids were harvested with 1 mg/mL Dispase II (Life Technologies) and pelleted by spinning at 500xg, 4 min, 4°C. Organoids were trypsinized to single cells using TrypsinEDTA (Sigma-Aldrich), trypsin activity was blocked with trypsin inhibitor (Sigma-Aldrich) at a 1:1 ratio, and single cells were pelleted by spinning at 500xg, 4min, 4°C. To visualise dead cells on FACS, 1.5 µM DRAQ7 (Cell Signaling Technologies) was added to growth medium containing 10 µM Y-27632 (Gentaur). On FACS, alive cells with STAR expression being positive (pos) or negative (neg) were collected duplicates or triplicates (10,000-20,000 cells each). Samples were pelleted and snap-frozen until processed further.
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| Growth protocol |
Engineered human colorectal cancer organoids (APCKO/KO; KRASG12D/-; TP53KO/KO) were grown from single STAR+ cells for 5-12 days in Matrigel (Corning). Growth media was refreshed every 2-3 days. Growth media contained Advanced DMEM/F12 (Invitrogen), supplemented with 10 mM Hepes (Invitrogen), 1% GlutaMAX (Invitrogen), 1% Penicillin-Streptomycin (Lonza) as well as 1x B27 supplement (Invitrogen), 1.25 mM N-Acetyl-L-cysteine (Sigma-Aldrich), 10 mM Nicotinamide (Sigma-Aldrich), 500 nM A83-01 (Tocris), and 10 µM SB202190 (Gentaur).
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using the RNeasy Mini RNA extraction kit (Qiagen, Cat. No. 74106) according to the manufacturer’s instructions with DNaseI treatment.
Up to 73ng RNA per sample was used for the preparation of the RNA sequencing libraries using the KAPA RNA HyperPrep Kit with RiboErase (HMR) (KAPA Biosystems). In short, oligo hybridisation and rRNA depletion, rRNA depletion cleanup, DNase digestion, DNase digestion cleanup, and RNA elution were performed according to protocol. Fragmentation and priming were performed at 94⁰C for 6:30 min. First strand synthesis, second strand synthesis, and A-tailing were performed according to protocol. For the adapter ligation, a 1.5μM stock was used (NextFlex DNA barcodes, Bioo Scientific). First and second post-ligation cleanup were performed according protocol. The library was amplified in 13 cycles and the subsequent cleanup was performed using a 0.8x bead-based cleanup. Library size was determined using the High Sensitivity DNA bioanalyzer (Agilent Technologies) and the library concentration was measured using the KAPA Library Quantification Kit (KAPA Biosystems). Sequencing was performed using an Illumina NextSeq 500, while 50‐bp paired‐end reads were generated.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
STAR+ cells were collected from 5-day-old AKP organoids for bulk RNA-seq.
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| Data processing |
Reads were aligned with Hisat2 (version 2.0.4) with enabled alignment tailoring for transcript assemblers.
Reads per gene were counted with htseq-count script (Hisat2 software suite, strandness disabled, identification attribute set to gene_id).
Genes with no reads in any of the samples were excluded from analysis.
Differential gene expression was performed with DESeq2 package.
Significantly changing genes were called after Walt test followed by correction for multiple testing (Benjamini-Hochberg method).
Data visualisation was performed upon normalisation, log2 transformation, and noise-stabilisation using the rlogTransformation function of the DESeq2 package.
Assembly: hg38
Supplementary files format and content: txt files containing transcript counts (PCR duplicates have been removed)
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| Submission date |
Jun 28, 2022 |
| Last update date |
Dec 01, 2023 |
| Contact name |
Maria Christine Heinz |
| E-mail(s) |
Maria.Puschhof@uni-heidelberg.de
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| Organization name |
University Medical Center Utrecht
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| Department |
Molecular Cancer Research
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| Lab |
Hugo Snippert
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| Street address |
Heidelberglaan 100
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| City |
Utrecht |
| ZIP/Postal code |
3584 CX |
| Country |
Netherlands |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE207070 |
Transcriptome and chromatin changes during CRC organoid outgrowth as proxy for metastatic outgrowth [bulkRNAseq_AKP] |
| GSE207071 |
Transcriptome and chromatin changes during CRC organoid outgrowth as proxy for metastatic outgrowth |
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| Relations |
| BioSample |
SAMN29397034 |
| SRA |
SRX15924355 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6276716_TPO3-hColonOrganoids-StarPosNeg-RNAseq-TPO3-pp-6-RNA-3-16792_rmDupCounts.txt.gz |
144.7 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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