title: RNA-seq of Pseudomonas aeruginosa treated with Melastoma dodecandrum Lour and antibiotics
design: RNA-seq of Pseudomonas aeruginosa treated with Melastoma dodecandrum Lour and antibiotics
library_construction_protocol: Collection of PAO1 cells following treatment by compounds Overnight cultures were sub-cultured and grown to mid-log phase to OD=0.4. Dissolved in antibiotic solution in Mueller-Hinton Broth (MHB) to a final concentration of OD=0.2, MIC=1X, and final volume of 500uL. Samples incubated at 37C statically for 30 mins. 1mL of RNA protect reagent added to 500uL of sample. Cells were lysed via treatment with TE buffer containing 15mg/mL lysozyme for 10 mins with frequent vortexing, following manufacturer's instructions Directional mRNA library preparation (no ribosomal RNA depletion). Following fragmentation, cDNA was synthesized using random hexamer primers and then the dUTPs were replaced with dTTPs. Finally, the following was performed: end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification.