GSM8223907: Mtb, control, rep 2; Mycobacterium tuberculosis; RNA-Seq
DFO (0.1 mg/ml) and control were harvested at day 5 post-addition of Deferoxamine by centrifuging the Mtb cultures at 3,500 g for 10 minutes at room temperature. The cell pellet was washed with Tris-EDTA to remove extra media and the pellet was suspended in Trizol reagent (1 ml). The samples were subjected to bead beating (4 cycles, 45 sec each) after adding zirconia beads (~200 μl, 0.1 mm) at 6.5 m/s speed with incubation of 30 sec on ice after each cycle. Chloroform (0.2 ml) was added to the samples and after briefly shaking the tubes, the samples were incubated on ice (for 5 min) and centrifuged at 13,400 g, 4ºC for 20 min. Upper aqueous layer was transferred to a fresh tube and RNA was precipitated by adding isopropyl alcohol (0.5 ml). The samples were then incubated at -80ºC for 20 min and subsequently centrifuged for 10 min at 13,400 g and 4ºC. The purified RNA pellet was washed with 75% ethanol and resuspended in nuclease-free water. NEBNext Ultra RNA library prep kit