title: GSM2415042: WT 2; Vibrio harveyi; RNA-Seq
library_construction_protocol: Cells grown to OD600=1.0 were pelleted by centrifuging and snap frozen in N2. Total RNA was isolated from cells using a Trizol/cholorform extraction protocol from a previous study (Rutherford et al., 2011). RNA was cleaned up using an Rneasy kit (Qiagen) cDNA libraries were prepared from total RNA by the Indiana University Center for Genomics and Bioinformatics. mRNA was enriched from one microgram total RNA using RiboZero rRNA Removal (Bacteria) Kit (Illumina, Inc., San Diego, CA, USA). rRNA-depleted mRNA samples were purified by Agencourt AMPure® XP beads (Beckman Coulter, Indianapolis IN, USA) and viewed using Agilent High Sensitivity RNA ScreenTape (Agilent Technologies, Inc., Santa Clara, CA, USA). Double stranded cDNA synthesis was performed using Illumina TruSeq Stranded mRNA Library Prep Kit in accordance with the manufacturer's standard protocol. Enriched mRNA was fragmented using divalent cations via incubation for 8 min at 94oC. The first strand of cDNA was synthesized by reverse transcription using random-sequence primers. Second-strand synthesis was performed by subsituting dTTP with dUTP followed by digestion using Uracil-DNA-Glycosylase (UDG). A-tailing and adapter ligation was performed  followed by PCR amplification for 12 cycles using polymerase provided in kit. This step generated the second strand of cDNA and completed the addition of Illumina adapter sequences incorporating a user-defined barcode. The amplified libraries were purified using Agencourt AMPure® XP beads. Quality and quantity were assessed using an Agilent DNA1000 ScreenTape (Agilent Technologies, Inc., Santa Clara, CA, USA) and Picogreen assay kit (Invitrogen, Carlsbad, California, USA), respectively. Libraries were made single-stranded and diluted to 1.8pM, as per Illumina manufacturer's instructions. Cluster generation and sequencing was performed on an Illumina NextSeq500 using NextSeq 500/550 High Output v2 kit (75 cycles). Single-end 80bp sequencing was performed on an Illumina NextSeq500 using NextSeq 500/550 High Output v2 kit (75 cycles).
attributes.tag: GEO Accession
attributes.value: GSM2415042