GSM6602725: Ca_6hr_2; Mycobacterium tuberculosis; RNA-Seq
RNA was isolated using published protocols (Rustad TR, Harrell MI, Liao R, Sherman DR (2008) The Enduring Hypoxic Response of Mycobacterium tuberculosis. PLoS ONE 3(1): e1502). In summary, cell pellets were resuspended in Trizol and lysed by bead beating for 30sec at 6m/s for three cycles with intermittent cooling on ice. Cell debris were pelleted at 20,000g for 1min, and the supernatant was transferred to a heavy phase lock gel tube containing 300 µl chloroform. The tubes were inverted for 2min and centrifuged at 20,000g for 5min. RNA in the aqueous phase was precipitated using 300 μl isopropanol and 300 μl high salt solution (0.8M Na citrate, 1.2M NaCl). RNA was purified using QIAGEN RNeasy kit and ribosomal RNA was depleted using the Ribo-Zero rRNA removal magnetic kit (Illumina) according to manufacturer's instructions. The cDNA library was generated using the NEBNext Ultra II RNA Library Prep Kit and each replicate was barcoded in the DNA library using the NEBNext Multiplex Oligos for Illumina. Libraries were quantified using the KAPA qPCR quantification kit, pooled, and sequenced at the University of Washington Northwest Genomics Center with the Illumina NextSeq 500 High Output v2 Kit.