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| Item Type: | Dataset |
|---|---|
| Title: | Sensitive dissection of a genomic regulatory landscape using bulk and targeted single-cell activation [NuCaptureC] |
| Creators Name: | Vučićević, Dubravka, Hsu, C.W., Lopez Zepeda, L.S., Burkert, M., Hirsekorn, A., Bilić, I., Kastelić, N., Landthaler, M., Lacadie, S.A. and Ohler, U. |
| Abstract: | ORGANISM: Homo sapiens. EXPERIMENT TYPE: Other. SUMMARY: Transcriptional enhancers are non-coding DNA elements that regulate gene transcription in a temporal and tissue-specific manner. Despite advances in computational and experimental methods, identifying enhancers and their target genes essential for specific biological processes remains challenging. Determining target genes for enhancers is also complex and often relies on indirect, low-resolution, and/or assumptive methodologies. To identify and functionally perturb enhancers at their endogenous sites without altering their sequence, we performed a pooled tiling CRISPR activation (CRISPRa) screen surrounding PHOX2B, a master regulator of neuronal cell fate and a key player in neuroblastoma development. This screen allowed the identification of CRISPRa- responsive elements (CaREs) that alter cellular growth within the 2 Mb genomic region. To determine CaRE target genes, we developed TESLA-seq (TargEted- SingLe- cell- Activation), which combines CRISPRa screening with targeted single-cell RNA-sequencing, and identified functional CaRE-target gene pairs. While most TESLA-revealed CaRE-gene relationships involved neuroblastoma-related regulatory elements already active in the system, we found many CaREs and target connections normally active only in other tissue types or with no previous evidence and induced out of context by CRISPRa. This highlights the power of TESLA-seq to reveal gene regulatory networks active outside of a given experimental system. OVERALL DESIGN: nuCapture C was performed according to (Downes et al., 2022). Single-stranded DNA probes were obtained from IDT as an xGen Lockdown Pool and are listed in the manuscript. Paired-end sequencing (2x150nt) was performed on a NextSeq 500/550 using a HighOutput v2 Kit for 300 cycles (Illumina #FC-420-1004, discontinued). |
| Source: | Gene Epression Omnibus (GEO) |
| Publisher: | NCBI |
| Date: | 1 June 2025 |
| Official Publication: | https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE274257 |
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