GSE155708
GSE GEORobust single-cell discovery of RNA targets of RNA binding proteins and ribosomes [NP]
Relations
Summary
RNA binding proteins (RBPs) are critical regulators of gene expression and RNA processing that are required for gene function. Yet, the dynamics of RBP regulation in single cells is unknown. To address this gap in understanding, we developed STAMP (Surveying Targets by APOBEC Mediated Profiling), which efficiently detects RBP RNA interactions. STAMP does not rely on UV crosslinking or immunoprecipitation and, when coupled with single cell capture, can identify RBP and cell type specific RNA protein interactions for multiple RBPs and cell types in single pooled experiments. Pairing STAMP with long read sequencing also yields RBP target sites for full length isoforms. Finally, conducting STAMP using small ribosomal subunits (RiboSTAMP) allows analysis of transcriptome wide ribosome association in single cells. STAMP enables the study of RBP RNA interactomes and translational landscapes with unprecedented cellular resolution.
Overall Design
This data was generated using a strategy for immunoprecipitation-free detection of protein-RNA interactions by fusion of RNA binding proteins and ribosomal subunits to the C-to-U RNA editing enzyme APOBEC1 and quantification of interaction-driven edits by sequencing, to allow interaction detection on long-read and single-cell platforms.
Analysis (11 steps)
View Data Processing- Raw reads were aligned to hg19 (genomic) or ENSEMBL (cDNA) reference using Minimap2 with the following parameters: "-ax splice -uf -k14".
- Aligned reads were then passed through Bcftools mpileup command with a "-Q 5" parameter.
- The pileup files were then filtered for G & C reference positions.
- For direct-RNA data the strand was inferred based on mapping directionality.
- For cDNA alignments the strand as assumed to be positive.
- For direct-cDNA reads aligned to hg19 sites were intersected with an annotation file and strand was assigned based on gene directionality with ambiguous strands being removed.
- Sites were then further filtered for C positions based on strand.
- Confidence scores were calculated using manual implementation of SAILOR with a modification rate of 5%.
Supplementary Files (1)
GEO Samples (4)
Dataset Citations (2)
Linked Publications (2)
Data Files (5)
| Accession | File Name | Stored Type | Output Type | Mapping Assembly | Size | Download | |
|---|---|---|---|---|---|---|---|
| — | APOBEC_cDNA_all_pass.fastq.gz | RNA-Seq | 7.7 GB | link | |||
| — | APOBEC_dRNA.fastq.gz | RNA-Seq | 545.2 MB | link | |||
| — | RBFOX2_cDNA_all_pass.fastq.gz | RNA-Seq | 21.1 GB | link | |||
| — | RBFOX2_dRNA.fastq.gz | RNA-Seq | 1.2 GB | link | |||
| — | SRR12389276.lite | RNA-Seq | 21.1 GB | link |