GSE205536
GSE GEO Potentially relatedMultiplexed transcriptome discovery of RNA binding protein binding sites by antibody-barcode eCLIP
Relations
Summary
UV cross-linking and immunoprecipitation (CLIP) methodologies enable the identification of RNA binding sites of RNA-binding proteins (RBPs). Despite improvements in the library preparation of RNA fragments, the current enhanced CLIP (eCLIP) protocol still requires ~4 days of hands-on time and lacks the ability to scale. We present a new method termed antibody barcode CLIP (ABC) that utilizes DNA-barcoded antibodies to multiplex CLIP detection methods. We demonstrate the scalability and simplicity of ABC by performing CLIP on multiple RBPs simultaneously, minimizing sample-to-sample variation, and maintaining the same material requirement for a single eCLIP experiment.
Overall Design
ABC(Antibody barcoded eCLIP) is perform as singleplex (RBFOX2, SLBP) or multiplex (for 10 RBPs)
Analysis (13 steps)
View Data Processing- Data is processed in a similar way to standard eCLIP pipeline, except for a few adjustments to ABCâs multiplex design and library structure.
- UMI are extracted using umitools 1.0.0 and adaptors are removed using cutadapt 2.8, then fastqs are demultiplexed based on the 5nt barcode sequence using fastx toolkit.
- Since ABC is sequenced on the reverse strand, we reverse complement sequences.
- Alignment to the repetitive regions removes multi-mapped reads, then aligned to the genomic sequences using STAR 2.7.6.
- The pipeline is available at https://github.com/algaebrown/oligoCLIP.git
- Given the genome alignment, we subsequently follow the steps to do peak call using CLIPper, then rank the peaks comparing to âbackgroundsâ, either SMInput (eCLIP), RNA-seq (ABC), or to other multiplex libraries (internal normalization).
- CLIPper uses nearby regions (+/- 500 b.p.) to estimate the background, followed by a Poisson distribution to assess the significance of a peak, whereas ânormalizationâ takes into account of another background library.
- In the eCLIP protocol, the SMInput library is prepared the same way as the IP library, which estimates the background crosslinking rate of other RBPs with similar size ranges as well as the RNA expression level and all other bias in library preparation.
Supplementary Files (1)
GEO Samples (9)
Dataset Citations (1)
Potentially Related Publications (1)
This accession was text-mined from the PMC full text of these publications. It may be referenced for comparison or reused from another study rather than generated for them.
Data Files (9)
| Accession | File Name | Stored Type | Output Type | Mapping Assembly | Size | Download | |
|---|---|---|---|---|---|---|---|
| — | HEK293_RBFOX2_rep1.fastq.gz | OTHER | 806.2 MB | link | |||
| — | HEK293_RBFOX2_rep2.fastq.gz | OTHER | 487.3 MB | link | |||
| — | HEK293_rep1.fastq.gz | OTHER | 7.2 GB | link | |||
| — | HEK293_rep2.fastq.gz | OTHER | 5.1 GB | link | |||
| — | K562_rep1.fastq.gz | OTHER | 4.8 GB | link | |||
| — | K562_rep4.fastq.gz | OTHER | 5.0 GB | link | |||
| — | K562_rep6.fastq.gz | OTHER | 3.8 GB | link | |||
| — | K562_SLBP_rep1.fastq.gz | OTHER | 911.3 MB | link | |||
| — | K562_SLBP_rep2.fastq.gz | OTHER | 382.0 MB | link |