title: GSM7698032: Spn delta lctO delta spxB (3); Streptococcus pneumoniae; RNA-Seq
library_construction_protocol: S. pneumoniae cultures (OD600nm 1.0) were aliquoted to 0.5 ml and treated with 1.0 ml RNA protect (Qiagen) for 5 min at RT. Bacterial cells were then collected by centrifugation at 13.000 rpm for 5 min and stored at -80°C.  RNA was isolated using the miRNAse kit (Qiagen), and the protocol was modified as follows. The collected bacterial pellets were thawed and washed with SET buffer [50 mM NaCl, 5 mM EDTA, and 30 mM Tris-HCl (pH 7.0)]. Subsequently, cells were centrifuged at 16.000 g for 3 min, and pellets were resuspended in Tris-HCl (pH 6.5) containing 40 U of SUPERase, 0.2 mg of proteinase K (Ambion), 50 mg/ml lysozyme (Sigma), and 25 U of mutanolysin (Sigma). After incubation for 30 min at 37 °C and 350 rpm, QIAzole (Qiagen) was added for 3 min at RT. Then, 0.2 volume chloroform was added followed by incubation for 3 min at RT. After centrifugation (16.000 x g at 4°C for 15 min)Tthe upper aqueous phase containing the RNA was transferred to a new collection tube. 1.5 volume ethanol (100%) was added to the sample and mixed. The aqueous phase and ethanol were transferred to miRNeasy Kit column (Qiagen) and treated according to the user's manual. Total RNA was depleted of ribosomal RNA with NEBNext® rRNA Depletion Kit v2 (Bacteria) (New England Biolabs, Ipswich, MA, USA) and Agencourt RNAClean XP beads (Beckman Coulter, Pasadena, CA, USA). Fragmentation and library preparation was then performed according to the manufacturer's instructions using NEBNext® Ultra II Directional RNA Library Prep Kit for Illumina. To facilitate multiplex sequencing, NEBNext® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs) (New England Biolabs, Ipswich, MA, USA) was used to barcode the reads.  Libraries were further purified with Agencourt AMPure XP Reagent (Beckman Coulter, Pasadena, CA, USA).