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Sample GSM6276698 Query DataSets for GSM6276698
Status Public on Dec 01, 2023
Title RNA_AKPS_Day5_STAR-pos-to-pos_3
Sample type SRA
 
Source name AKPS organoid
Organism Homo sapiens
Characteristics tissue: AKPS organoid
cell type: Colorectal cancer
time: Day 5
star day 0: pos
star day 5/12: pos
technical replicate: 3
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.
Growth protocol Engineered human colorectal cancer organoids (APCKO/KO; KRASG12D/-; TP53KO/KO; SMAD4 KO/KO) were grown from single STAR+ or 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).
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.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
 
Description STAR+ cells derived from 5-day-old AKPS organoids were sorted and STAR+ cells were collected for bulk RNA-seq.
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)
 
Submission date Jun 28, 2022
Last update date Dec 01, 2023
Contact name Maria Christine Heinz
E-mail(s) Maria.Puschhof@uni-heidelberg.de
Organization name University Medical Center Utrecht
Department Molecular Cancer Research
Lab Hugo Snippert
Street address Heidelberglaan 100
City Utrecht
ZIP/Postal code 3584 CX
Country Netherlands
 
Platform ID GPL18573
Series (2)
GSE207069 Transcriptome and chromatin changes during CRC organoid outgrowth as proxy for metastatic outgrowth [bulkRNAseq_AKPS]
GSE207071 Transcriptome and chromatin changes during CRC organoid outgrowth as proxy for metastatic outgrowth
Relations
BioSample SAMN29397020
SRA SRX15924369

Supplementary file Size Download File type/resource
GSM6276698_TPO4-hColonOrganoids-StarPosNeg-RNAseq-TPO4-pp-5-RNA-3-16468_rmDupCounts.txt.gz 148.8 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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