GSM5694060: T4delta_ccpA_CDMN_Exp_2_20210119; Streptococcus pneumoniae TIGR4;             RNA-Seq
GSM5694060
Samples were stored in RNAprotect Bacteria Reagent                     (Qiagen), and stored at -80C. On the day of RNA extractions, samples were thawed                     and spun down to discard the supernatant. Pellets were then incubated in 100 µL                     of lysis buffer (10 µL of mutanolysin, 20 µL of proteinase K,30 µL of lysozyme,                     40 µL of TE buffer) for 10 minutes. Pellets were mechanically disrupted in 600                     µL RLT buffer (RNeasy Mini Kit, Qiagen) containing 1% b-mercaptoethanol, using a                     motorized pestle for 30 seconds. Samples were then spun through a Qiashredder                     followed by the addition of 590 µL of 80% ethanol, and RNA was captured on the                     RNeasy Mini Kit columns with DNase treatment on column (Qiagen protocol),                     followed by additional DNase treatment in solution. Extracted RNA was                     quantitated using a Bioanalyzer (Agilent). Ribosomal RNA was depleted using the                     NEBNext rRNA Depletion Kit Bacteria (Catalog #: E7850S) for Gram-positive +                     Gram-negative species. 300 bp-insert RNA-seq Illumina libraries were constructed                     using 0.01 – 2.0 µg of enriched mRNA that was fragmented then used for synthesis                     of strand-specific cDNA using the NEBNext Ultra II Directional RNA Library Prep                     Kit (NEB #E7103). The cDNA was purified between enzymatic reactions and the size                     selection of the library performed with AMPure SpriSelect Beads (Beckman Coulter                     Genomics). The titer and size of the libraries were assessed on the LabChip GX                     (Perkin Elmer) and with the Library Quantification Kit (Kapa                     Biosciences).