GSM7061726: S.islandicus.REY15A.cbp1.Cappable-seq.replicate2; Sulfolobus islandicus REY15A; OTHER
15 ml cell culture was rapidly mixed with 30 ml 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) with an initial resuspension buffer volume of 200 µl following the protocol for small RNAs (20-200 nt length). Library preparation and deep sequencing was conducted at Vertis Biotechnologie (Germany). In brief, 5'-triphosporylated RNA was capped with 3'-desthiobiotin-TEG-GTP (NEB)) using the Vaccinia virus Capping enzyme (NEB) and biotinylated RNA species were subsequently enriched by affinity purification using streptavidin beads yielding 0.6 to 1.3% of the sRNA preparation. The eluted RNA was poly-adenylated using E. coli Poly(A) polymerase and 5'-ends were converted to mono-phosphates by incubation with RNA 5' Pyrophosphohydrolase (NEB). Subsequently, an RNA adapter was ligated to the newly formed 5'-monophosphate structures. First-strand cDNA synthesis was performed using an oligo(dT)-adapter primer and the M- MLV reverse transcriptase at 42 ºC. The resulting cDNA was finally PCR-amplified (12 cycles) with TruSeq Dual Index sequencing primers (Illumina).