GSM6429728: AEP1 (WT) + preQ1-L1, small RNAs, - rRNA, IP, rep3 (Expt2); Schizosaccharomyces pombe; RIP-Seq
Small RNAs were isolated using the PureLink™ miRNA Isolation Kit (Invitrogen) according to the manufacturer's instructions. Ribosomal RNA depletion of 5S and 5.8S rRNA was achieved using reverse complementary oligonucleotides with a 5'-biotin tag as described before (Kraus et al. 2019). 8 µg small RNAs were used for hybridization with 100 µM oligonucleotides in 10 µL of formamide, 2.5 µL of 20 x SSC, 5 µL of 0.005 M EDTA, pH 8 with the following thermocycling: 80 °C for 5 min, ramp down to 25 °C at intervals of 5 °C per minute. The reaction was stopped with the addition of 2 µl RNAseOut ans 50 µl of 1 x SCC containing 20% formamide. Removal of rRNA/oligonucleotide hybrids was performed using DynabeadsTM MyOneTM Streptavidin C1 (ThermoFisher) according to the manufacturer's instructions. Library preparation of immunoprecipitated RNAs for deep sequencing was performed using the NEBNext Small RNA Library Prep Set for Illumina (Multiplex Compatible; New England Biolabs) according to the manufacturer's protocols.