Explore Workflows

Cross-Linking ImmunoPrecipitation ================================= `CLIP` (`cross-linking immunoprecipitation`) is a method used in molecular biology that combines UV cross-linking with immunoprecipitation in order to analyse protein interactions with RNA or to precisely locate RNA modifications (e.g. m6A). (Uhl|Houwaart|Corrado|Wright|Backofen|2017)(Ule|Jensen|Ruggiu|Mele|2003)(Sugimoto|König|Hussain|Zupan|2012)(Zhang|Darnell|2011) (Ke| Alemu| Mertens| Gantman|2015) CLIP-based techniques can be used to map RNA binding protein binding sites or RNA modification sites (Ke| Alemu| Mertens| Gantman|2015)(Ke| Pandya-Jones| Saito| Fak|2017) of interest on a genome-wide scale, thereby increasing the understanding of post-transcriptional regulatory networks. The identification of sites where RNA-binding proteins (RNABPs) interact with target RNAs opens the door to understanding the vast complexity of RNA regulation. UV cross-linking and immunoprecipitation (CLIP) is a transformative technology in which RNAs purified from _in vivo_ cross-linked RNA-protein complexes are sequenced to reveal footprints of RNABP:RNA contacts. CLIP combined with high-throughput sequencing (HITS-CLIP) is a generalizable strategy to produce transcriptome-wide maps of RNA binding with higher accuracy and resolution than standard RNA immunoprecipitation (RIP) profiling or purely computational approaches. The application of CLIP to Argonaute proteins has expanded the utility of this approach to mapping binding sites for microRNAs and other small regulatory RNAs. Finally, recent advances in data analysis take advantage of cross-link–induced mutation sites (CIMS) to refine RNA-binding maps to single-nucleotide resolution. Once IP conditions are established, HITS-CLIP takes ~8 d to prepare RNA for sequencing. Established pipelines for data analysis, including those for CIMS, take 3–4 d. Workflow -------- CLIP begins with the in-vivo cross-linking of RNA-protein complexes using ultraviolet light (UV). Upon UV exposure, covalent bonds are formed between proteins and nucleic acids that are in close proximity. (Darnell|2012) The cross-linked cells are then lysed, and the protein of interest is isolated via immunoprecipitation. In order to allow for sequence specific priming of reverse transcription, RNA adapters are ligated to the 3' ends, while radiolabeled phosphates are transferred to the 5' ends of the RNA fragments. The RNA-protein complexes are then separated from free RNA using gel electrophoresis and membrane transfer. Proteinase K digestion is then performed in order to remove protein from the RNA-protein complexes. This step leaves a peptide at the cross-link site, allowing for the identification of the cross-linked nucleotide. (König| McGlincy| Ule|2012) After ligating RNA linkers to the RNA 5' ends, cDNA is synthesized via RT-PCR. High-throughput sequencing is then used to generate reads containing distinct barcodes that identify the last cDNA nucleotide. Interaction sites can be identified by mapping the reads back to the transcriptome.

https://github.com/datirium/workflows.git

Path: workflows/clipseq-se.cwl

Branch/Commit ID: 4360fb2e778ecee42e5f78f83b78c65ab3a2b1df

https://github.com/ProteinsWebTeam/ebi-metagenomics-cwl.git

Path: tools/SSU-from-tablehits.cwl

Branch/Commit ID: b6d3aaf3fa6695061208c6cdca3d7881cc45400d

https://github.com/common-workflow-language/cwltool.git

Path: cwltool/schemas/v1.0/v1.0/js-expr-req-wf.cwl

Branch/Commit ID: 227f35a5ed50c423afba2353871950aa61d58872

Packed ID: wf

https://github.com/ProteinsWebTeam/ebi-metagenomics-cwl.git

Path: workflows/16S_taxonomic_analysis.cwl

Branch/Commit ID: 85155424fa5654526517369be2fa479a7d4d90de

https://github.com/EBI-Metagenomics/ebi-metagenomics-cwl.git

Path: tools/tRNA_selection.cwl

Branch/Commit ID: 3f85843d4a6debdabe96bc800bf2a4efdcda1ef3

https://github.com/proteinswebteam/ebi-metagenomics-cwl.git

Path: workflows/emg-core-analysis-v4.cwl

Branch/Commit ID: 7bb76f33bf40b5cd2604001cac46f967a209c47f

https://github.com/EBI-Metagenomics/ebi-metagenomics-cwl.git

Path: tools/collate_unique_SSU_headers.cwl

Branch/Commit ID: 583307878ab83c5845c897f03db920ae8e1929e2

https://github.com/ProteinsWebTeam/ebi-metagenomics-cwl.git

Path: workflows/16S_taxonomic_analysis.cwl

Branch/Commit ID: a8abd0e66de7b5ffe24cfe7f39d7027103c6d3b4

https://github.com/ncbi/pgap.git

Path: task_types/tt_align_sort_sa.cwl

Branch/Commit ID: 2353ee2550529ca5b0705c94b32022a21713db18

https://github.com/common-workflow-language/cwltool.git

Path: tests/wf/scatter-wf4.cwl

Branch/Commit ID: 75271e2a0887d47cca4077b60dd51ac763c09b63

Packed ID: main