title: Biofilm microbial community composition at deepsea hydrothermal vents, in the Pacific Ocean
design: Total RNA was extracted from the xx g of biofilm biomass in RNA later and resuspended in solution I (50 mM glucose, 10 mM EDTA, 25 mM tris-HCl pH 8.0) and 75 L of 0.5 M EDTA. The samples were subjected to three freeze/thaw cycles, after which 100 L of lysozyme solution (4 mg/mL lysozyme in Solution I) was added, and samples were set to shake for 15 minutes. Afterwards, 50 L 10% SDS was added to each sample. Samples were extracted twice with 800 L phenol pH 4.3 and once using 800 L phenol-cholorform-isoamyl alchohol (25:24:1) pH 6.7/buffer pH 8.0. RNA was precipitated by adding 0.1 volumes of 3.0 M Na-acetate and 1 mL of 100% ice-cold ethanol at -20C overnight. The samples were centrifuged at 4C for 30 minutes, supernantant was removed, and samples were washed with 1 mL 80% ethanol, centrifuged, dried and resuspended in 50 L ddH20. DNase-treated RNA was reversed transcribed to single-stranded cDNA using SuperScript III First-Strand Synthesis System with SuperScript III Reverse Transcriptase primed with random hexamers (Invitrogen, Carlsbad, CA, USA). Samples were subsequently stored at -20C until further analyses.