GSM9422067: k2; Mycolicibacterium smegmatis; RNA-Seq
M. smegmatis and its ΔkuΔligD were cultured in 7H9 medium to an OD600 of 2.0. Bacterial cells were collected by centrifugation and resuspended in fresh 7H9 medium to an OD600 of 1.0. The cultures were then infected with SWU1 phage at an MOI of 10, followed by a 30 minutes adsorption period at 37℃. Subsequent incubation was carried out at 37°C with shaking at 200 rpm for 30 and 180 minutes. At each time point, bacteria were harvested by centrifugation, and total RNA was extracted for RNA-seq analysis. RNA was fragmented and then used as template for first-strand cDNA synthesis with reverse transcriptase and random hexamer primers, followed by second-strand cDNA synthesis using RNase H and DNA polymerase I. The resulting double-stranded cDNA fragments underwent end repair, addition of a single “A” base, and adapter ligation. Adaptor-ligated fragments were selected using AMPure XP beads and amplified by PCR to generate the final cDNA library. Transcriptome sequencing was performed by Wuhan Feisha Gene Company using an Illumina platform.