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| Status |
Public on Jan 01, 2024 |
| Title |
MethNet: a robust approach to identify regulatory hubs and their distal targets in cancer [Perturb-seq] |
| Organism |
Homo sapiens |
| Experiment type |
Other
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| Summary |
We present MethNet, a pipeline that integrates large-scale DNA methylation and gene expression data across multiple cancers, to uncover novel cis regulatory elements (CREs) in a 1Mb region around every promoter in the genome. MethNet identifies highly ranked CREs, referred to as ‘hubs’, which contribute to the regulation of multiple genes and strongly affect patient survival. Promoter-capture Hi-C confirmed that highly ranked associations involve physical interactions between CREs and their gene targets, and CRISPRi based scRNA Perturb-seq validated the functional impact of CREs. Thus, MethNet represents a valuable resource for unraveling complex mechanisms underlying gene expression, and for prioritizing the verification of predicted non-coding disease hotspots.
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| Overall design |
CloneTracker XP CRISPR Barcode pooled lentiviral libraries expressing barcoded sgRNAs, the puromycin selection gene as well as an RFP reporter were purchased from Cellecta®. The A549 cells were transduced with lentiviruses expressing dCas9-KRAB-MECP2 plasmids as described in Yeo et al. Cells were then grown in DMEM medium (Gibco/Invitrogen) + 10% FBS + 100 units/ml penicillin + 100 μg/ml streptomycin + 5% CO2 at 37℃. The 248 sgRNAs targeting high-confidence MethNet hubs and 5 non-targeting sgRNAs (negative controls) were designed using CRISPick for the Human GRCh37 (hg19) assembly using parameter settings: CRISPRi, SpyoCas9/Chen (2013) tracrRNA (Suppl. Table 1). The lentiviral libraries were transduced into the A549-dCAS9-KRAB-MeCP2 cells according to Cellecta® protocols. Briefly, 105 cells/well were seeded into a 6 well-plate. The optimal MOI of viral particles (to reach ~30-40% of infected cells) were added. On day 3, 2 ug/ml of puromycin was added, resulting in >90% transduced cell selection as confirmed by cytofluorometry using RFP (Figure S7). Cells were expanded under puromycin selection until day 14. For scRNA-seq using the 10X Genomics technology, 25,000 cells were harvested. For optimal multiplet detection and optimal signal-to-noise ratios, cells were hashtagged using 5 cell multiplexing oligos purchased from 10X Genomics. The sequencing library was, then, prepared using the Chromium Next GEM Single Cell 3ʹ Kit according to the manufacturer’s protocol and sequenced by NYU Langone's Genome Technology Center.
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| Contributor(s) |
Sakellaropoulos T, Jiang G, Meyn P, Heguy A, Tsirigos A, Do C, Skok J |
| Citation(s) |
37577603, 39025865 |
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| Submission date |
Jun 30, 2023 |
| Last update date |
Aug 09, 2024 |
| Contact name |
Theodoros Sakellaropoulos |
| E-mail(s) |
theodoros.sakellaropoulos@nyulangone.org
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| Organization name |
NYU Langone
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| Street address |
550 1st Avenue
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| City |
New York |
| ZIP/Postal code |
10016 |
| Country |
USA |
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| Platforms (1) |
| GPL24676 |
Illumina NovaSeq 6000 (Homo sapiens) |
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| Samples (3) |
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| This SubSeries is part of SuperSeries: |
| GSE236305 |
MethNet: a robust approach to identify regulatory hubs and their distal targets in cancer |
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| Relations |
| BioProject |
PRJNA989650 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE236304_barcodes.tsv.gz |
117.6 Kb |
(ftp)(http) |
TSV |
| GSE236304_feature_reference.csv.gz |
4.5 Kb |
(ftp)(http) |
CSV |
| GSE236304_features.tsv.gz |
328.5 Kb |
(ftp)(http) |
TSV |
| GSE236304_filtered_feature_bc_matrix.h5 |
193.0 Mb |
(ftp)(http) |
H5 |
| GSE236304_matrix.mtx.gz |
535.5 Mb |
(ftp)(http) |
MTX |
| GSE236304_protospacer_umi_thresholds.csv.gz |
1.4 Kb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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