Alternative cDNA library prep protocols
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We’ve described a comparison of the variant options for the library preparation protocols in our .
What is the feasibility of doing RNA library prep using a commercially available kit (NEB Ultra, or Illumina Tru-seq), after RNA extraction following proteinase digestion? In this way, the isolated RNA will be treated as a normal RNA sample and random hexamer primers would be used for reverse-transcription.
The pro is that you can use an established kit, so possibly less optimisation needed. The cons are several. Some are described in our review (greater specificity with on-bead ligation, the capacity for non-radioactive visualisation in irCLIP, etc). In addition, I’m not aware of a kit that would ligate the adapter to cDNA to allow amplification of truncated cDNAs. So when using kits, data will be similar to HITS-CLIP, and restricted to readthrough cDNAs. Also, CLIP RNAs are often too short to allow priming with random hexamers. So all in all, use of kits may work in some cases, but could lead to biased data with limited resolution.