RIP-seq experiment to evaluate the role of methylation of the spliceosome component LSM4 in the regulation of alternative splicing

The RIP-seq experiment was designed to look for the direct targets of LSM4 U6snRNP component. For this plants were grown in Murashige and Skoog (MS) plates in continuous light (LL) for 12 days and were vacuum-infiltrated with 1% formaldehyde for 15 min followed by quenching with 125 mM glycine. A whole-cell extract was prepared in RIP-lysis buffer. The extract was pre-cleared with Sepharose beads and subjected to immunoprecipitation with GFP-Trap beads (Chromotek). After extensive washing with RIP washing buffer, co-precipitated RNAs were extracted with Trizol (Invitrogen) and treated with DNase (Promega). Libraries were prepared and sequenced at the Max Planck-Genome-Centre Cologne (MP-GC).