GSE155726
GSE GEORobust single-cell discovery of RNA targets of RNA binding proteins and ribosomes [scRNA-seq]
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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
Cells expressing RBP-APOBEC1 fusions for C-to-U editing of RBP target-RNAs were processed through the Chromium Single Cell Gene Expression Solution using the Chromium Single Cell 3â Gel Bead, Chip, 3â Library and 3â Feature Barcode Library Kits v3 (10X Genomics) as per the manufacturerâs protocol. The following fusion expressions were processed for a target of 10,000 cells/ experiment: transient transfection of control- and RBFOX2-APOBEC1 (STAMP) in HEK293T and separately in neural progenitor cells (NPC), independent stable inductions of control- and RBFOX2:TIA1-STAMP (cell mixture, capture sequence barcoded), and RPS2-STAMP.
Analysis (12 steps)
View Data Processing- Alignment, cell barcode processing, umis processing, abundance measurements: cellranger count (version 3.0.2)
- MD tags were added to alignments with samtools calmd --threads 15 -rb possorted_genome_bam.bam refdata-cellranger-hg19-3.0.0/fasta/genome.fa > possorted_genome_bam_MD.bam
- Reads were split based on the CB:Z tag, resulting in one BAM file per barcode.
- Edits were called on each "split-BAM" file using SAILOR (http://github.com/yeolab/sailor) using default parameters and dbSNP (v147) to remove known SNPs.
- Coverage was generated from filtered BAM files (intermediates from SAILOR) for each cell barcode, using the following commands from BedTools (v2.27.1). :
- bedtools genomecov -split -strand - -g refdata-cellranger-hg19-3.0.0/star/chrNameLength.txt -bg -ibam fwd.bam > fwd.bg
- bedtools sort -I fwd.bg > fwd.sorted.bg
- bedGraphToBigWig fwd.sorted.bg refdata-cellranger-hg19-3.0.0/star/chrNameLength.txt fwd.sorted.bw
Supplementary Files (1)
GEO Samples (5)
Dataset Citations (2)
Linked Publications (2)
Data Files (5)
| Accession | File Name | Stored Type | Output Type | Mapping Assembly | Size | Download | |
|---|---|---|---|---|---|---|---|
| — | control_STAMP_possorted_genome_bam.bam | RNA-Seq | 12.0 GB | link | |||
| — | NPC_HEK_control_STAMP_possorted_genome_bam.bam | RNA-Seq | 8.6 GB | link | |||
| — | NPC_HEK_RBFOX2_STAMP_possorted_genome_bam.bam | RNA-Seq | 8.6 GB | link | |||
| — | RBP_STAMP_possorted_genome_bam.bam | RNA-Seq | 18.6 GB | link | |||
| — | RPS2_STAMP_possorted_genome_bam.bam | RNA-Seq | 12.3 GB | link |