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König J, Zarnack K, Luscombe NM, Ule J. Protein-RNA interactions: new genomic technologies and perspectives. Nat Rev Genet. 2011;13(2):77-83.
This variation
Each of these protocols can be executed in numerous ways, and the details of implementation (such as the specific enzyme used during RNase digestion in a PAR-CLIP experiment) can affect results in different ways. However, all of these experiments share the structural properties of A) producing a FASTQ file which is then aligned to the genome/transcriptome to produce a BAM file, and 2) producing these files for an IP sample and an Input sample, Igg sample, or both as controls.
Important Protocol-Data Structure Relationships
| Variable | RIP-Seq | HITS-CLIP | PAR-CLIP |
|---|---|---|---|
| Cross-linking | None | With 254 nm radiation; Introduces somewhat less predictable mutations | After incubation with 4-thiouridine; With 3565 nm radiation; Introduces predominantly T to C transitions in sequencing data |
| Lysis | Very gentle to avoid disrupting RNP complexes
| Often uses RNases, sonication, or both to disrupt large masses of cross-linked RNPs | Similar to HITS-CLIP, with different optimization of biochemical details (e.g. enzyme choice and concentration) |
| RNA Selection | Can use poly-A selection and/or ribo-depletion; Size selection only after fragmentation since targeted RNAs may be of variable length; No RNP electrophoresis | No poly-A selection; Ribo-depletion possible; Population selection by excision from gel relative to native protein; Involves radioactive RNP electrophoresis | Similar to HITS-CLIP, with different optimization of biochemical details |
| Sequencing | Long, paired-end reads often used; Data spans the whole length of captured RNA molecules with some bias towards the ends | Short, single-end reads often used; Data piles up on the cross-linked sites with many mutations | Short, single-end reads often used, though paired-end reads can be employed on short fragments to provide extra confidence in T to C transitions |