title: RNAseq of C. trachomatis: Vector_control__+ATc_R1
design: L929 cells in a 6-well tissue culture plate with tetracycline-free RPMI media were infected with the pL2-tetO ctcC ATPase only construct or the pL2-tetO vector control. The infection was induced with a final ATc concentration of 10 ng/mL at 16 hpi. A relatively small window of induction was chosen to reduce the pleiotropic effect of over-activating 54.  At 20 hpi, the infection was harvested as described in the previous section using TriZol and subsequent chloroform RNA extraction. As per the procedure for RNA-sequencing intracellular bacteria by Marsh, Humphrys, and Myers, following TURBO DNase treatment (Thermo Fisher, Waltham, MA) and purification using the RNeasy kit (Qiagen, Hilden, Germany), both host and chlamydial ribosomal RNA was depleted using the Ribo-Zero Magnetic Core Kits (Illumina, San Diego, CA) specific for Human/Mouse/Rat rRNA and Gram-negative bacteria rRNA [77]. A final purification step was performed using the RNeasy Mini kit (Qiagen, Hilden, Germany). rRNA depletion was assessed by running samples on a 2% agarose gel. RNA quality was assessed using Qubit quantification and TapeStation gel analysis. The RNA library was made using the NEBNext Ultra II Direactional RNA Library Prep Kit for Illumina (New England BioLabs, Ipswich, MA) and was sequenced with NextSeq 550 High Output Single Read 50 bp Sequencing (NX-HO-SR50).