title: GSM2563959: Azotobacter vinelandii wild-type (DJ) strain 15 hours grown in standard B Medium Supplemented with 20 mM NH4 [AV.RNA.42]; Azotobacter vinelandii; RNA-Seq
library_construction_protocol: Samples for RNA isolation were flash frozen in liquid nitrogen and stored until needed. RNA was isolated by resuspending frozen cells in 1 mL of TRIzol reagent (Invitrogen, Grand Island, NY) and samples were vortexed intermittently for several minutes until fully dissolved. Following this, 200 mL of chloroform was added, vortexed, and then centrifuged at 12,000 g for 2 minutes. The upper phase was removed, and further purified using the Direct-zol RNA miniprep kit (Zymo Research, Irvine, CA). RNA was eluted and treated to remove background DNA following the manufacturer's directions using the RNase-free DNase I Set (Zymo Research, Irvine, CA) in a total volume of 100 mL for 10 min at room temp, and then suspended in 300 mL of TRIzol and again isolated using the Direct-zol miniprep kit. Isolated RNA quantity was measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA). Ribosomal and transfer RNAs were removed from the total isolated RNA using the Ribo-Zero kit for Gram-Negative bacteria (Illumina Inc., San Diego, CA). Final mRNA fractions were concentrated using the RNA Clean & ConcentratorTM-5 (Zymo Research, Irvine, CA). Samples were then sequenced by HiSeq 2500 Rapid Mode with 100-bp paired-end reads. Raw data from the RNA sequencing experiments was run through the rnaseq-pipeline (tophat/hisat) for the analysis of high-throughput sequence datasets through the University of Minnesota Supercomputing Institute. The pipeline utilized FastQC, Trimmomatic, and Bowtie 2. Cuffquant and Cuffnorm used to calculate fragments per kilobase of exon per million fragments mapped (FPKM) values for each gene. Further comparisons were made by exporting this data to Excel (Microsoft, Redmond, WA) for a further comparison of differences in transcriptional levels.
attributes.tag: GEO Accession
attributes.value: GSM2563959