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| Status |
Public on Feb 06, 2025 |
| Title |
Comparison of RNA quality in fresh and RAID-fixed cells with drug-induced replication stress |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
The underlying mechanisms that influence cancer cells' sensitivity to replication stress are impeded by the analysis of bulk-samples which neglect tumor heterogeneity and fail to accurately interpret cell cycle mediated resistance. Here, by combining intracellular immunostaining and RNA-sequencing of single cells, we characterized the transcriptomes of subsets of cells with oncogenic RAS that exhibit low levels of RS even when challenged with a CHK1 inhibitor and gemcitabine.
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| Overall design |
All samples consist of human RPE cell lines harboring the Tet Repressor, FUCCI4 system, fluorescently tagged 53BP1 and HRASG12V, which are described in Segeren et al 2022. Cells with doxycycline-inducible oncogenic HRASG12V were treated with replication stress inducing drugs, gemcitabine + prexasertib (referred to as GP). In this experiment, cells were not treated with doxycycline, and thus have no expression of HRASG12V. Cells in S and G2 phase were selected based on expression of the FUCCI marker Geminin1-110. Cells were either freshly sorted prior to scRNA sequencing (fresh) or they were reversibly fixed, intracelularly stained with yH2AX, and decrosslinked prior to scRNA sequencing (RAID). Fresh cells and RAID-fixed cells were each sorted into 384-well plates, with half of each plate containing veh-treated cells and the other half containing GP-treated cells. After sorting, scRNA sequencing was performed to correlate the degree of yH2AX staining to transcriptional profiles of individual cells.
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| Contributor(s) |
Segeren HA, Wierenga KA, Westendorp B, Riemers FM |
| Citation(s) |
40009518 |
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| Submission date |
Feb 20, 2024 |
| Last update date |
May 08, 2025 |
| Contact name |
Frank Riemers |
| E-mail(s) |
f.m.riemers@uu.nl
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| Organization name |
Utrecht University
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| Street address |
Uppsalalaan 8
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| City |
Utrecht |
| ZIP/Postal code |
3584 CT |
| Country |
Netherlands |
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| Platforms (1) |
| GPL18573 |
Illumina NextSeq 500 (Homo sapiens) |
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| Samples (2) |
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| Relations |
| BioProject |
PRJNA1078308 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE256134_RAW.tar |
4.4 Mb |
(http)(custom) |
TAR (of TSV) |
| GSE256134_celseq2_bc.tsv.gz |
2.1 Kb |
(ftp)(http) |
TSV |
| GSE256134_fresh_dsp_count_data.tsv.gz |
3.4 Mb |
(ftp)(http) |
TSV |
| GSE256134_fresh_dsp_meta_data.tsv.gz |
46.3 Kb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
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