GSM5418748: Spombe csRNA-seq rep1; Schizosaccharomyces pombe; RNA-Seq
Trizol extraction after tissue homogenisation For RNA-Seq, strand-specific, paired-end libraries were prepared from total RNA by ribosomal depletion using the Yeast Ribo-Zero rRNA Removal Kit (Illumina) and then using the TruSeq Stranded total RNA-Seq kit (Illumina) according to manufacturer's instructions. Then 100 bases were sequenced from both ends using a HiSeq 4000 according to the manufacturer's instructions (Illumina).    csRNA-seq was performed as described in (Duttke et al. 2019). Small RNAs of ~20-60 nt were size selected from 0.4-2 µg of total RNA by denaturing gel electrophoresis. A 10% input sample was taken aside and the remainder enriched for 5'-capped RNAs. Monophosphorylated RNAs were selectively degraded by Terminator 5´-Phosphate-Dependent Exonuclease (Lucigen) and RNAs were 5'dephosporylation by quickCIP (NEB). Input (sRNA) and csRNA-seq libraries were prepared as described in (Hetzel et al. 2016) using RppH (NEB) and the NEBNext Small RNA Library Prep kit, amplified for 14 cycles and sequenced SE75 on the Illumina NextSeq 500. csRNA-seq, sRNA-seq, total RNA-seq