title: RNA-Seq of Tenacibaculum discolor sv11:  T.discolor nrps-prom GFP cultured in MMB medium to OD600 0.4 - Replicate 2
design: Wild type and mutant T. discolor sv11 strains were cultured in three biological replicates under the specified conditions for each experiment. We cultured T. discolor::aclA supplemented with C7-ACL or DMSO in MMB, and performed RNA sequencing on cells harvested 12 h after treatment. T. discolor sv11 NRPS-GFP reporter strain was cultivated and cells at different growth stages were collected when optical density (600 nm) of cultures reached 0.03, 0.2 and 0.4. Cells were collected by centrifugation in an Eppendorf centrifuge 5810R at 4 C and 15000 rpm for 2 min. Cell pellets were immediately processed for RNA isolation using the RNAeasy Minikit (Qiagen), followed by a DNAse treatment using TurboTM DNAse (Thermo Fisher). The integrity of the bacterial total RNA was first examined on a Bioanalyzer 2100 using the RNA 6000 Pico chip (Agilent Technologies, CA). Messenger RNA (mRNA) was enriched from the samples using the Ribo-Cop rRNA Depletion Kit for Bacteria (Lexogen Inc, NH). Enriched mRNA samples were further fragmented and converted to cDNA and Illumina sequencing library using the PrepX RNA-seq library preparation protocol on the automated Apollo 324TM NGS Library Prep System (Takara Bio, CA). A unique DNA barcode was incorporated into each library during this process to enable sample multiplexing in each pool. The prepared RNA-seq libraries were examined on Agilent Bioanalyzer DNA High Sensitivity chips for size distribution, quantified by Qubit fluorometer (Invitrogen, CA), and pooled at equal molar amount. The library pools were denatured and sequenced on an Illumina NovaSeq 6000 using the S Prime flowcells as paired-end 150 nt reads according to the manufacturers protocol. Raw sequencing reads were filtered by Illumina NovaSeq Control Software, only the Pass-Filter (PF) reads were de-multiplexed and used for further analysis.