GSM7026769: RSNC137C-3; Sphingomonas melonis TY; RNA-Seq
Total bacterial samples were collected (A600 = 0.6 at the time of collected), mixed with 0.2 volumes of stop-mix (95% ethanol and 5% phenol, v/v) and snap-frozen in liquid nitrogen. Pellets were resuspended in 100 μl lysozyme solution (15 mg/ml lysozyme in TE buffer, pH = 8.0). Total RNA was isolated with Omega-E.Z.N.A.® Bacterial RNA Kit. Briefly, RNA degradation and contamination were monitored on 1% agarose gels. The integrity of RNA sample was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, USA). Sequencing libraries were generated from rRNA-depleted RNA using the RiboMinus Bacteria 2.0 Transcriptome Isolation Kit (Thermo Scientific, USA) following the manufacturer's recommendations. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer's instructions. The library preparations were sequenced on an Illumina Novaseq platform and 150 bp paired-end reads were generated. Before data analysis, raw data were firstly processed through in-house perl scripts as a quality control. Demultiplexed and quality filtered reads were then aligned to S. melonis TY genome sequence using Bowtie2-2.2.3. HTSeq 0.6.1 was used to count the reads numbers mapped to each gene.