GSM9108680: ppe51_KO_3; Mycobacterium tuberculosis; RNA-Seq
The bacteria were harvested at the indicated timepoint and resuspended in Trizol, and lysed by bead beating for 30 seconds at 6 m/s for 3 cycles, with intermittent cooling on ice. Cell debris was pelleted at 20,000 g for 1 minute, and the supernatant was transferred to a heavy phase-lock gel tube containing 300 μL of chloroform. The tubes were inverted for 2 minutes and centrifuged at 20,000 g for 5 minutes. RNA in the aqueous phase was precipitated with 300 μL of isopropanol and 300 μL of high-salt solution (0.8 M sodium citrate, 1.2 M sodium chloride). RNA was purified using the QIAGEN RNeasy kit, and ribosomal RNA was depleted using a previously published protocol [Culviner, P. H. et al doi:10.1128/mBio.00010-20 (2020)]. Briefly, a biotinylated oligo mixture of 23S, 16S, and 5S was incubated with RNA to anneal to rRNA, followed by incubation with streptavidin beads. mRNA was purified from the supernatant using Ampure XP beads. 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.