GSE166404
GSE GEORobust single-cell discovery of RNA targets of RNA binding proteins and ribosomes [STAMP_pacbio]
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 yields RBP target sites in an isoform-specific manner. Finally, Ribo-STAMP leverages small ribosomal subunits to measure transcriptome-wide ribosome association in single cells. STAMP enables the study of RBP-RNA interactomes and translational landscapes with unprecedented cellular resolution.
Overall Design
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 (9 steps)
View Data Processing- The double stranded cDNA for each sample was converted to sequencing libraries as recommended (PacBio SMRTbell Express Template Prep Kit 2.0) but with separate barcoded adapters for each sample (PacBio Barcoded Overhang Adapter Kit).
- All of the samples were pooled in an equimolar fashion and sequenced on a SMRTcell 8M with the PacBio Sequel II instrument (2.0 chemistry/2.1 polymerase with 2 hour pre-extension and 30 hour movie times).
- After barcodes were demultiplexed, the initial data was used to re-balance the pooling by barcode counts before further sequencing.
- In total, the samples were sequenced over 5 SMRTcell 8M.
- The PacBio Sequel II system was used for all sample sequencing.
- Following sequencing, the circular consensus sequence (CCS) reads for each set of technical replicates were processed using the Isoseq v3 pipeline (https://github.com/PacificBiosciences/IsoSeq) to generate full-length non-concatamer reads in fasta format.
- For this step, software package lima v2.0.0 was used with parameters: --isoseq and --dump-clips.
- In addition, isoseq3 v3.4.0 refine was used with parameters: --require-polya.
Supplementary Files (1)
GEO Samples (2)
Dataset Citations (2)
Linked Publications (2)
Data Files (10)
| Accession | File Name | Stored Type | Output Type | Mapping Assembly | Size | Download | |
|---|---|---|---|---|---|---|---|
| — | CCS_Demux_5759_APO1_1_demultiplex.bc1001_BAK8A_OA__bc1… | OTHER | 32.0 MB | link | |||
| — | CCS_Demux_5759_APO1_2_demultiplex.bc1015_BAK8B_OA__bc1… | OTHER | 29.7 MB | link | |||
| — | CCS_Demux_5759_APO1_3_demultiplex.bc1016_BAK8B_OA__bc1… | OTHER | 20.7 MB | link | |||
| — | CCS_Demux_5779_APO1_1_demultiplex.bc1001_BAK8A_OA__bc1… | OTHER | 327.6 MB | link | |||
| — | CCS_Demux_6465_APO1_1_demultiplex.bc1001_BAK8A_OA__bc1… | OTHER | 37.6 MB | link | |||
| — | CCS_Demux_6465_APO1_2_demultiplex.bc1015_BAK8B_OA__bc1… | OTHER | 55.7 MB | link | |||
| — | CCS_Demux_6465_APO1_3_demultiplex.bc1016_BAK8B_OA__bc1… | OTHER | 37.0 MB | link | |||
| — | CCS_Demux_6499_APO1_1_demultiplex.bc1001_BAK8A_OA__bc1… | OTHER | 43.8 MB | link | |||
| — | CCS_Demux_6499_APO1_2_demultiplex.bc1015_BAK8B_OA__bc1… | OTHER | 34.3 MB | link | |||
| — | CCS_Demux_6499_APO1_3_demultiplex.bc1016_BAK8B_OA__bc1… | OTHER | 28.8 MB | link |