RNA-Seq of seawater: Offshore CEWAF Timepoint 0
Filtered seawater samples were homogenized for 2 min in TE buffer                 (1mM EDTA, 10mM Tris; pH 6.3) with ~1g of sterilized 0.1mm diameter zirconia/silica                 beads. After bead beating, crude extracts were amended with two volumes of chilled                 (4) denaturing buffer (4M guanidine thiocyanate, 50mM Tris, 10mM EDTA, 1% w/v                 N-lauroylsarcosine, 1% ?-mercaptoethanol) and pelleted via centrifugation (4500xg                 for 5 min at 4). The resulting supernatant was extracted with an equal volume of                 phenol:chloroform:isoamyl alcohol (25:24:1, pH 6.6), followed by a second extraction                 with chloroform:isoamyl alcohol (24:1). Nucleic acids were purified from these                 extracts via an overnight isopropanol precipitation with 3M sodium acetate (pH 6.0)                 and a subsequent 70% ethanol wash, followed by resuspension in 100 of TE buffer.                 Genomic DNA was eliminated from RNA samples with TURBO DNAse (Ambion) followed by                 purification with MEGAclear Transcription Clean-up kits (Ambion). The resulting                 purified total RNA extracts were processed with MICROBExpress kits (Ambion) to                 reduce the amount of 16S and 23S rRNA transcripts within the samples and then sent                 to the University of Delaware DNA Sequencing & Genotyping Center (Newark, DE,                 USA) for Illumina HiSeq 2500 sequencing (SINGLE-end 150bp                 reads).
SUB6082343
M3_T0_CEWAF_metaT