Solid-Support Directional (SSD) RNA-Seq as a Companion Method to CLIP-Seq
CLIP-Seq (Deep Sequencing after in vivo Crosslinking and Immunoprecipitation, HITS-CLIP) has emerged as a key method for the study of RNA-binding proteins (RBPs), as it can scrutinize the RNAs bound by an RBP in vivo, with minimum manipulation of biological samples. CLIP-Seq is best used to reveal changes of the RNA cargo of an RBP and differences on binding patterns of the bound RNAs in living cells in different genetic backgrounds or after experimental treatment, rather than simply identifying RNA species. It is therefore crucial that a reference of the steady state levels of the RNAs present in the samples used for the CLIP-Seq experiment is included in the bioinformatic analysis. A simple directional RNA-Seq method was developed that uses the same oligonucleotides and the same PCR amplification steps as our CLIP-Seq method, which therefore can be analyzed using the same bioinformatic pipeline as the CLIP-Seq data. This greatly simplifies and streamlines the analysis process, and at the same time reduces the chances of protocol-specific artifacts and biases interfering with data interpretation. Some considerations on ways to integrate CLIP-Seq and RNA-Seq analyses are also provided herein.