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| 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 365 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 |
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As you have already learned, after getting your raw sequencing data, most pipelines will evaluate the quality of the raw data, followed by filtering/pre-processing and alignment. In the case of RIP-Seq experiments, these steps with tools like the Tuxedo pipeline (for example) can often use knowledge that depends on the protocol steps described above, such as the distribution of fragment sizes or the tolerance of the aligner for mutations (e.g. mismatches). What you always end up with, though, are several SAM/BAM files, preferably one for each Input, Igg, and IP. Let's take a look at some high-level differences between some datasets that suggest obvious protocol differences.
First, below is a snapshot of what a clear RIP-Seq peak might look like, derived from some Iyer Lab data: