GSE155728
GSE GEORobust single-cell discovery of RNA targets of RNA binding proteins and ribosomes [tia1_eclip]
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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
Crosslinking and immunoprecipitation of TIA1 bound RNA and sequencing from 2 technical replicates.
Analysis (21 steps)
View Data Processing- Sequenced reads were removed of inline barcodes and reformatted to include randomers in read headers with eclipdemux (v0.0.1).
- Args: --length 10
- Reads were then trimmed with cutadapt (1.14).
- Args: --match-read-wildcards -O 1 --times 1 -e 0.1 --quality-cutoff 6 -m 18 -g g_adapters.fasta -A A_adapters.fasta -a a_adapters.fasta (fasta sequences generated from parsebarcodes.sh within the eclip 0.1.5+ pipeline)
- Reads were then trimmed once more with cutadapt (1.9.1) to remove double-ligation events.
- Args: --match-read-wildcards -O 5 --times 1 -e 0.1 --quality-cutoff 6 -m 18 -A A_adapters.fasta (fasta sequences generated from parsebarcodes.sh within the eclip 0.1.5+ pipeline)
- Trimmed reads were then mapped with STAR (2.4.0i) against a human-specific repeat element database (RepBase 18.05).
- Args: --runThreadN 16 '--genomeDir human_repbase '--readFilesIn path/to/read1 path/to/read2 '--outFileNamePrefix out_prefix '--outReadsUnmapped Fastx '--outSAMtype BAM Unsorted '--outSAMattributes All '--outSAMunmapped Within '--outSAMattrRGline ID:foo '--outFilterType BySJout '--outFilterMultimapNmax 30 '--outFilterMultimapScoreRange 1 '--outFilterScoreMin 10 '--alignEndsType EndToEnd