GSM6265688: Alginate oligosaccharide planktonic replicate 1; Vibrio splendidus; RNA-Seq
A Qiagen RNeasy kit was used to isolate total RNA, following manufacturer's protocol, except for the following modifications: cells were resuspended in 15 mg/mL lysozyme in TE buffer, and incubated for room temperature for 30 min, prior to addition of buffer RLT. Samples were lysed by mechanical disruption using lysing matrix B (MPBio, Santa Ana, CA). Samples in lysing matrix shaken in a homogenizer (MPBio) for 10X 30 second intervals, taking care to keep samples from heating up. Samples were treated with DNase digestion using a Turbo DNA free DNAse kit (Ambion, Austin, TX), following manufacturer's instructions. The integrity and purity of total RNA was assessed running a sample on an Agilent 4200 Tapestation in a HS RNA screen tape (Agilent, Santa Clara, CA). Depletion of rRNA was accomplished using the Ribominus Yeast and Bacteria transcriptome isolation kit (Invitrogen, Waltham, MA) and included ethanol precipitation with glycogen to concentrate remaining mRNA. Ethanol precipitated samples were resuspended in FPF sample buffer from the TruSeq stranded mRNA-Seq kit reagents (Illumina, San Diego, CA). mRNA abundance was quantified using a Quant-iT RNA Assay kit (Invitrogen) on a Tecan Spark plate reader equipped with a monochrometer, following manufacturer's instructions (Tecan, Männendorf, Switzeland). The quality of the mRNA was further assessed using an Agilent HS tape. We analyzed resulting samples on a Bioanalyzer to confirm mRNA depletion, and concentrated samples using ethanol precipitation with glycogen. Samples were screened for quality by requiring an RNA integrity number (RIN) or more than 8. mRNA remaining from rRNA depletion of 2-10 µg total RNA was used as template to create sequencing libraries. Sequencing libraries were prepared using an Illumina TruSeq Stranded mRNA library preparation kit (Illumina). Libraries were assessed for quality using screen tapes and fluorescence-based plate reader assays prior to pooling.