GSM7312100: bphOP1_KO_pN-bphOP1_far-red, rep 2; Pseudomonas syringae pv. syringae B728a; RNA-Seq
Immediately after treatment, 0.5 mL of each culture sample was added to 1 mL of RNAprotect (Qiagen) and vortexed for 5 sec. Following a 5-min incubation at room temperature, the cells from the two independent cultures for a single strain x treatment combination were combined and centrifuged at 5,000 x g for 10 min. The supernatant was decanted, and the cell pellet was stored at -70°C. For each strain, this process was repeated on six separate days. The cell pellets for a given strain x treatment combination collected on two separate days were thawed on ice and combined, and this pool was considered one biological replicate. In this manner, each strain x treatment combination was represented by three biological replicates, each of which contained cells derived from four independently exposed cultures. Total RNA was extracted using a RNeasy Mini Kit (Qiagen) with on-column Dnase I treatment, with one biological replicate for each strain x treatment combination extracted at the same time. Library construction and sequencing were performed by BGI Genomics (China). For each sample, total RNA was depleted for ribosomal RNA using an Epicentre Ribo-Zero Magnetic Kit (Bacteria), fragmented, and then used for RNA-seq library construction using a TruSeq RNA Sample Prep Kit v2 (Illumina). Libraries were assessed for quality using a 2100 Bioanalyzer, quantified using real-time quantitative PCR, and sequenced on an Illumina HiSeq 4000 with 100-bp paired-end reads.