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| Item Type: | Dataset |
|---|---|
| Title: | Sensitive dissection of a genomic regulatory landscape using bulk and targeted single-cell activation [Chromatin RNA-seq] |
| 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: Cellular fractionation was performed in SH-SY5Y cell line according to (Conrad and Ørom, 2017). Two replicates of chromatin RNA were extracted using Trizol and Direct-zol RNA MiniPrep Kit (Zymo Research #R2052) according to the manufacturer’s instructions. The library was prepared using NEXTflex Rapid Directional qRNA-Seq Kit (BiooScientific #NOVA-5130-01D) according to the manufacturer’s instructions and paired-end sequencing (2x75nt) was performed on a NextSeq 500/550 using a HighOutput v2 Kit for 150 cycles. |
| Source: | Gene Epression Omnibus (GEO) |
| Publisher: | NCBI |
| Date: | 11 September 2025 |
| Official Publication: | https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE274258 |
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