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| Status |
Public on Dec 01, 2023 |
| Title |
ATAC_AKPS_Day5_STAR-pos-to-pos_2 |
| Sample type |
SRA |
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| Source name |
AKPS organoid
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| 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: 2
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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 frozen in Recovery Cell Culture Freezing Medium (Gibco) prior to processing.
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| 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).
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| 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).
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
STAR+ cells derived from 5-day-old AKPS organoids were sorted and STAR+ cells were collected for bulk ATAC-seq.
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| 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)
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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) |
| 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 |
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| Relations |
| BioSample |
SAMN29397086 |
| SRA |
SRX15924590 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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