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Sample GSM6276669 Query DataSets for GSM6276669
Status Public on Dec 01, 2023
Title ATAC_AKPS_Day5_STAR-neg-to-neg_1
Sample type SRA
 
Source name AKPS organoid
Organism Homo sapiens
Characteristics tissue: AKPS organoid
cell type: Colorectal cancer
time: Day 5
star day 0: neg
star day 5/12: neg
technical replicate: 1
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 frozen in Recovery Cell Culture Freezing Medium (Gibco) prior to processing.
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 genomic DNA
Extraction protocol Cells were lysed and DNA was extracted after performing the transposition reaction with a Tn5 enzyme for 30 min at 37°C. Subsequently, the reaction was stopped through addition of 44 mM EDTA, 131 mM NaCl, 0.3 % SDS, and 600 µg/mL proteinase K. DNA was extracted using the Quiagen MinElute PCR Purification Kit. For further details, please refer to (Lindeboom et al., 2018).
Transposed DNA fragments were amplified using Custom Nextera PCR Primers 1 and 2 together with 10 µl DNA input. Subsequent clean-up was performed using reverse 0.65x SPRI beads (AMPure). An Illumina NextSeq 500 was used for sequencing of 50 bp paired-end reads. For further details, please refer to (Lindeboom et al., 2018).
 
Library strategy ATAC-seq
Library source genomic
Library selection other
Instrument model Illumina NextSeq 500
 
Description STAR- cells derived from 5-day-old AKPS organoids were sorted and STAR- cells were collected for bulk ATAC-seq.
Data processing Reads were aligned with BWA mem.
PCR duplicates were removed with picard tools version 1.129.
Reads were further filtered for a quality score of >= 1 and for not mapping to the mitochondrial chromosome.
Accesible sites were identified in each sample with macs2 using a Q value of 0.001. A union of all identified peaks was used for subsequent analyses.
Differential site accessibility was performed with DESeq2 package.
Significantly accessible sites were called after Wald 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: hg19
Supplementary files format and content: Single txt file containing read counts per peak of all samples (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)
GSE207068 Transcriptome and chromatin changes during CRC organoid outgrowth as proxy for metastatic outgrowth [bulkATACseq_AKPS]
GSE207071 Transcriptome and chromatin changes during CRC organoid outgrowth as proxy for metastatic outgrowth
Relations
BioSample SAMN29397093
SRA SRX15924583

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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