GSE205536

Multiplexed transcriptome discovery of RNA binding protein binding sites by antibody-barcode eCLIP

1. Data is processed in a similar way to standard eCLIP pipeline, except for a few adjustments to ABC’s multiplex design and library structure.
2. 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.
3. Since ABC is sequenced on the reverse strand, we reverse complement sequences.
4. Alignment to the repetitive regions removes multi-mapped reads, then aligned to the genomic sequences using STAR 2.7.6.
5. The pipeline is available at https://github.com/algaebrown/oligoCLIP.git
6. Given the genome alignment, we subsequently follow the steps to do peak call using CLIPper, then rank the peaks comparing to “backgrounds”, either SMInput (eCLIP), RNA-seq (ABC), or to other multiplex libraries (internal normalization).
7. CLIPper uses nearby regions (+/- 500 b.p.) to estimate the background, followed by a Poisson distribution to assess the significance of a peak, whereas “normalization” takes into account of another background library.
8. 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)

Dataset Citations (1)

Linked Publications (3)