GSM5233504: 13_MSMEG_ΔmadR_C; Mycolicibacterium smegmatis; RNA-Seq
The resuspended cells were transferred to a tube containing Lysing Matrix B (MP Biomedicals, Santa Ana, CA) and vigorously shaken at max speed for 30 s in a FastPrep 120 homogenizer (MP Biomedicals) three times. Tubes were centrifuged for 1 min (max speed), then 66 μL of 3 M sodium acetate pH 5.2 added and mixed well. Acid phenol (pH 4.2) was added at 726 μL and tubes were inverted to mix well (~60 times). Samples were incubated at 65° C for 5 min, inverting tubes to mix samples every 30 s. Then, centrifuged at 14000 rpm for 5 min and upper aqueous phase was transferred to a new tube. 3M sodium acetate (pH 5.2) was added at 1/10th volume along with 3x volumes of 100% ethanol. Sample was mixed well and incubated at -20° C for 1 hr or overnight. Following incubation, samples were centrifuged at 14000 rpm for 30 min at 4° C, ethanol was discarded and 500 μL of 70% ethanol was added. Samples were centrifuged again at 14000 rpm for 10 min at 4° C, supernatant discarded, and any residual ethanol removed using pipet. Pellet was allowed to air dry, resuspended in 30-40 μL of RNase free water and quantified by Nanodrop (Thermo Scientific). This was followed by in solution genomic DNA digestion using RQ1 Dnase (Promega) following manufacturer's recommendation. RNA libraries were prepared for sequencing using standard Illumina protocols