title: GSM6131802: 4M, ovoid cells; Mycolicibacterium smegmatis MC2 155; RNA-Seq
library_construction_protocol: Active and dormant (ovoid) cultures were harvested by centrifugation at 7000 rpm for 15 min and washed 3 times with an ice-cold PBSm buffer containing (per liter) 8 g NaCl, 0.2 g MgCl2, and 0.24 g Na2HPO4 (pH 7.0). The bacterial pellet was re-suspended in the same buffer containing phenylmethanesulfonyl fluoride (PMSF) then lysed by using zirconium beads on a bead beater homogenizer (MP Biomedicals FastPrep-24) for 30 sec, three times. The bacterial lysate was centrifuged at 13000 rpm for 10 min at 4 °C. The supernatant was separated into membrane and cytosolic fractions using ultracentrifugation at 105000g and 4 °C for 2 h. The membrane fraction was washed with PBSm using ultracentrifugation. For RNA isolation the pellet was re-suspended in ice-cold 100 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer (pH 8.0) containing 5 mM EDTA and the zwitterionic detergent CHAPS (3-(3-cholamidopropyl) dimethylammonio-1-propanesulfonate hydrate (2%) (w/v)). Total RNA waw isolated by phenol-chloroform extraction as described previously [Rustad TR, Roberts DM, Liao RP, Sherman DR. Isolation of mycobacterial RNA. Methods Mol Biol. 2009;465:13–21.] After isolation RNA was treated with Turbo DNase (Life Technologies, USA) to remove traces of genomic DNA. Amounts and purity of RNA were determined spectrophotometrically; integrity of RNA was assessed in 1% agarose gel. Sequencing libraries were generated using the resulting RNA and NEBNext Ultra II Directional RNA Library Prep Kit (NEB, USA) according to the manufacturers' protocol.