GSM3990103: RIP using strain containing no tags; Schizosaccharomyces pombe; RIP-Seq
Cells were fixed with 1% formaldehyde at room temperature for 20 minutes. Cells were lysed and were sonicated using bioruptor. Immunoprecipitations were performed using GFP antibodies (ab290). Reverse crosslinking was performed overnight at 65˚C and protein was degraded using proteinase K. RNAase free DNAase I (Thermo Fisher, am2222) was used to degrade DNA fragments and RNA was purified using phenol-chloroform and ethanol precipitation. NEB Ultra Directional RNA Library Prep kit (NEB, E7420S) was used according to manufacturer's recommendation. Libraries were analyzed using an Agilent 4200 TapeStation system (Agilent) and sequenced on the Illumina NextSeq 500 platform.