GSM7987475: BCG_wildtype_A_S25; Mycobacterium tuberculosis variant bovis BCG; RNA-Seq
Cells in RNA later were transferred to a tube containing Lysing Matrix B and vigorously shaken at max speed for 30 s in a FastPrep 120 homogenizer  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 14,000 rpm for 5 min and upper aqueous phase was transferred to a new tube. 3 M 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 14,000 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 quality was analyzed in a 2100 Bioanalyzer system. Total RNA samples were depleted of ribosomal RNA using the Illumina Ribo-Zero Bacteria rRNA Removal Kit. Samples were prepared with Illumina TrueSeq Stranded mRNA HT library preparation kit and sequenced on the NextSeq 2000 instrument with NextSeq P2 (200 cycles) kit.