GSM7925569: S.islandicus.REY15A.cbp1.RNA-seq.replicate2; Sulfolobus islandicus REY15A; RNA-Seq
Cell cultures were rapidly mixed with 2 volumes  pre-cooled RNAprotect Bacteria Reagent (Qiagen) placed in an ice bath. Cells were harvested by centrifugation (5 min at 4000 x g at 4 ºC). Pellets were immediately subjected to RNA isolation using the mirVana miRNA isolation kit (Ambion) following the protocol for total RNA. Ribosomal RNA was removed using the Pan-Archaea RNA-seq riboPool kit (siTOOLS biotech) and the RNA was subsequentially fragmented using Ambion RNA Fragmentation Reagents (Thermo Fisher) according to manufacturer's protocol. Fragments in the size range of 17-200 nt were subsequently purified using RNA Clean & Concentrator-5 kit (Zymo Research). Purified RNA fragments were treated to remove 3´ end blocking groups (in particular 2',3'-cyclic phosphate from crRNAs) using 10 U T4 polynucleotide kinase (NEB) for 1h 30 min at 37°C in  70 mM Tris/HCl pH 6.5, 10 mM MgCl2, 1 mM DTT, 20 U SUPERaseIn RNase Inhibitor (Ambion) in 20 µl reactions. To phosphorylate the 5´-ends, additional 10 U polynucleotide kinase (NEB) in presence of 1 mM ATP, 10 U SUPERaseIn RNase Inhibitor (Ambion), 1x T4 PNK buffer (NEB) were added to the reactions in a total reaction volume of 50 μl and the reactions were incubated for 40 min at 37°C. Finally, the treated RNA fragments were ethanol-precipitated, resuspended in 6 μl of H2O and converted to Illumina sequencing libraries using NEBNext Small RNA Library Prep Set for Illumina kit (NEB) with 12 cycles of PCR-amplification.