Identification of the RNA targets of RBM5 and transcriptome analysis of the brain of wild-type and R6/2 (Huntington's disease) mice

Using the Cross-linking immuno-precipitation (CLIP) methodology combined with high throughput sequencing analysis, we have identified the RNA targets of the RNA-binding protein RBM5 in the brain of wild-type and R6/2 (Huntington's disease model) adult mice. In parallel, transcriptome analysis was performed by RNA-sequencing using brain samples from the same animals. Mouse brains were harvested from three wild-type and three R6/2 12-weeks old male mice. Each brain was longitudinally bisected into two equivalent halves. Both halves were gently dissociated and homogenized. One half was exposed to UV254 for cross-linking and processed for CLIP and high throughput sequencing (CLIP-Seq) following a protocol previously published (PMID: 24407355). Briefly, after the first steps including gentle lysis and controlled RNA fragmentation, the lysate was divided into three equal fractions and each fraction was incubated with magnetic beads coupled with rabbit anti-RBM5 antibodies (AbR; ref# HPA018011, Sigma), mouse anti-RBM5 antibodies (AbM; ref# sc-515419, Santa Cruz) or non-specific antibodies (IgG) before the rest of the protocol was performed. Isolated RNAs were then processed for library preparation and high throughput sequencing (single-end reads, 150bp; Illumina MiSeq), resulting in 18 raw fastq files (NFO120-A to NFO120-R) and one xlsx processed file (CLIP-Seq_processed-data). Total RNA was extracted from a small portion of the non-cross-linked half of the brain and processed for RNA-Sequencing (paired-end reads, 2 x75bp; Illumina NextSeq500) according to standard protocol, and resulting in 48 raw fastq files (eight for each sample: NFO136-2 to NFO136-9) and one xlsx processed file (RNA-Seq_processed-data). For each experiment – CLIP-Seq and RNA-Seq – all samples were processed in parallel.