RNA seq of Deinococcus radiodurans
D. radiodurans WT, khpA, and khpB strains was inoculated in TGY to a final OD600 of 0.8. 10 mL of cells were harvested by centrifugation for 10 min at 4,000 g and 4C. The cells were then mixed with 1 mL TRIzol reagent (Invitrogen) and lysed through two rounds (100 s each) of beadbeating. 400 L chloroform was added to the TRIzol-dissolved lysate and mixed. After centrifugation for 15 min at 4C and 13, 000 rpm, RNA was precipitated with ice-cold 30:1 ethanol: 3M NaOAc (pH 5.2), washed with ice-cold 70% ethanol twice, and dissolved in 35 L RNase-free water. The RNA samples were then digested with DNase I (New England BioLabs) for 30 min at 37C and purified using the RNA Clean & Concentrator Kit (Zymo Research). The RNA samples were quantified and evaluated using Bioanalyzer and Qubit 4 Fluorometer (Invitrogen) before sequencing. Ribosomal RNA was depleted using the NEBNext rRNA Depletion Kit (Bacteria) (New England BioLabs). NEBNext Multiplex RNA Library Prep Set for Illumina (New England Biolabs) was used for library construction and sequencing was performed by the Genomic Sequencing and Analysis Facility at the University of Texas at Austin using the Illumina NovaSeq S1 single-end platform.