GSM5618022: pombe_WT_R_II; Schizosaccharomyces pombe; OTHER
The cell pellets were resuspended in 750μL of TES buffer (10mM Tris pH 7.5, 10mM EDTA pH 8, 0.5% SDS) and the total RNA was isolated by hot phenol extraction. Rhodamine labeling was performed as described in our manuscript. Briefly, 30μg of total RNA were resuspended in 1% sodium borohydride for 60min at 25°C. Mock samples (R-) were treated similarly but without sodium borohydride. The reduced RNA was then precipitated and the dried pellet was resuspended in 0.1M sodium formate pH 4. Rhodamine labeling was performed for 90min at 37°C in dark with 2.2mM of rhodamine chlorine. Labeled RNA was purified by phenol extraction. A synthetic dihydrouridilated RNA spike-in was added to the samples (1:1000) before ribodepletion of 5 μg of the RNA samples. Zinc-mediated fragmentation was performed at 70°C for 15min with 90-125 ng of ribodepleted RNA. Simultaneous DNA digestion and RNA extremities dephosphorylation were performed at 37°C for 30min with 2U of TURBO DNase and 3U of FastAP Thermosensitive Alkaline Phosphatase. 3'-end ligation of the RNA adapter was performed for 90min at 25°C with 39U of T4 RNA ligase. After ligation, 10pmol of DNA primer were added, the sample was denatured for 2min at 70°C and the reverse transcription was performed with Superscript III. The excess of DNA primer was subsequently digested with 4min incubation with 2μL of ExoSAP-IT. The 3'-end ligation of the cDNA with DNA adapter was performed overnight at 25°C with 48U of T4 RNA ligase. Nucleic acid clean-ups were performed between each step by using MyOne SILANE magnetic beads. 13 cycles od PCR amplification was performed in a 50μL-reaction by mixing cDNA, 1μM of barcoded PCR primer, 1μM of universal PCR primer and 25μL of NEBNext High- Fidelity 2X PCR Master Mix (NEB M0541). Finally, two successive rounds of DNA polymer size selection were performed by mixing PCR products with 1.2 and 1.1 volume of AMPure XP beads. The DNA library was quantified, examined on a DNA microchip and sequenced on Illumina HiSeq 2500 or Novaseq.