GSM8864073: ΔmutT4::mutT4 -RppH replicate 3; Mycobacterium tuberculosis; RNA-Seq
were centrifuged at 3,900 rpm for 7 min at 4 °C. The bacterial pellets were resuspended in 1 mL of TRI Reagent (Molecular Research Center, TR118) and transferred to bead-beating tubes (OPS Diagnostics 100 µm zirconium beads, PFMB 100-100-12). Cells were lysed with 3 cycles at 7 m/s, 30 seconds per cycle in a FastPrep-24 bead-beater (MP Biomedicals) and then 300 µL of chloroform was added. Samples were centrifuged 15,000 rpm for 15 min at 4 °C and RNA from the aqueous phase was purified using Direct-Zol RNA miniprep kit (Zymo Research, R2052) following the manufacturer's instructions. DNase treatment was performed on-column and the purified RNA was stored at -80 °C. rRNA was depleted as described in (Culviner, P.H., Guegler, C.K. and Laub, M.T. (2020) A simple, cost-effective, and robust method for rRNA depletion in RNA-sequencing studies. MBio, 11, 10.1128/mbio. 00010-00020.). Samples were then treated with E. coli RppH or mock-treated, and 5'-end directed RNAseq libraries were constructed as described (Martini, M.C., Sun, H. and Shell, S.S. (2021) RNA Sequencing for Transcript 5'-End Mapping in Mycobacteria. Methods Mol Biol, 2314, 513-531). A set of in-vitro transcribed, RppH-treated transcripts was added at the adapter ligation step to faciliate normalization of the data.