Illumina HiSeq 2000 sequencing; RIP-seq experiment to evaluate the role of methylation of the spliceosome component LSM4 in the regulation of alternative splicing
RIP-seq experiment to evaluate the role of methylation of the spliceosome component LSM4 in the regulation of alternative splicing
Plants grown in MS plates in continuous light (LL) for 12 days 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). RNAs were extracted with Trizol (Invitrogen) and treated with DNase (Promega).  Libraries were prepared by genome center at the Max Planck-Genome-Centre Cologne (MP-GC).