GSM8179737: Arabidopsis thaliana 6d seedling csRNA-seq r2; Arabidopsis thaliana; OTHER
A. thaliana seedlings tissue was ground using the Qiagen TissueLyser II, at 1/30s frequency for 1.5 min twice. RNA was purified using the Zymo Direct-zol RNA MiniPrep kit. csRNA-seq was performed as described in (S. H. Duttke et al. 2019). Small RNAs of ~20-60 nt were size selected from 0.4-3 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 1 hour incubation with Terminator 5´-Phosphate-Dependent Exonuclease (Lucigen). Subsequently, RNAs were 5'dephosporylated through 90 minutes incubation in total with thermostable QuickCIP (NEB) in which the samples were briefly heated to 75°C and quickly chilled on ice at the 60 minutes mark. 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 11-14 cycles. Total RNA-Seq Library Preparation Strand-specific, paired-end libraries were prepared from total RNA by ribosomal depletion using the Ribo-Zero Gold plant rRNA removal kit (Illumina, San Diego, CA). Samples were processed following the manufacturer's instructions.