Sample collection and storage: Subsurface sediment samples from the continental shelf of Peru, Ocean Drilling Program (ODP) Site 1229D (77° 57.4590′ W, 10° 58.5721′ S), were obtained during ODP Leg 201 on 6 March 2002. Careful precautions were taken to avoid contamination during the sampling process. For Integrated Ocean Drilling Program (IODP) cores, contamination tests were performed using perfluorocarbon tracers and fluorescent microspheres. Sediment samples were immediately frozen at −80 degrees C after sampling and stored at −80 degrees C until used for mRNA extractions in this study (10-year storage time at −80 degrees C).
RNA extraction and purification: Extraction of sub-seafloor RNA was performed according to the protocol described previously. In brief, RNA was extracted from 25 g of sediment using the FastRNA Pro Soil-Direct Kit (MP Biomedicals). It was necessary to scale up the volume of sediment that is typically extracted with the kit (~0.5 g) owing to the low biomass inherent to marine subsurface samples. All tubes, tips and disposables used were certified RNase free and all extraction procedures were performed in a laminar flow hood to reduce aerosol contamination by bacterial and fungal cells/spores. Five 15-ml Lysing Matrix E tubes (MP Biomedicals) were filled with 5 g sediment and 5 ml of Soil Lysis Solution (MP Biomedicals). Tubes were vortexed to suspend the sediment and Soil Lysis Solution was added to the tube leaving 1 ml of headspace. Tubes were then homogenized for 60 s on the FastPrep-24 homogenizer (MP Biomedicals) with a setting of 4.5. Contents were pooled into two 50-ml tubes and centrifuged for 30 min at 4000 r.p.m. (3220g) at room temperature (25 degrees C). Supernatants were combined in a new 50-ml tube and 1/10 volume of 2 M sodium acetate (pH 4.0) was added. An equal volume of phenol-chloroform (pH 6.5) was added and vortexed for 30 s, incubated for 5 min at room temperature, and spun at 4000 r.p.m. (3220g) for 20 min at 4 degrees C. The aqueous phase was transferred to a new 50-ml tube. Nucleic acids were precipitated by adding 2.5 and 1/10 volumes 100% ethanol and 3 M sodium acetate, respectively, and incubating overnight at −80 degrees C. The next day, tubes were spun at 4000 r.p.m. (3220g) for 60 min at 4 degrees C and the supernatant removed. Pellets were washed with 70% ethanol, spun for 15 min at 4 degrees C and air-dried. Dried pellets were resuspended with 0.25 ml RNase-free sterile water and combined into a new 1.5-ml tube. 1/10 volume of 2 M sodium acetate (pH 4.0) and an equal volume of phenol-chloroform (pH 6.5) were added, vortexed for 1 min and incubated for 5 min at room temperature. This was necessary to remove residual organic material (that is, humic acids) resulting from the rather large pellet/precipitate. After centrifuging at 14000 r.p.m. (20817g) for 10 min at 4 degrees C, the top phase was removed into a new 1.5-ml tube. 0.7 volumes of 100% isopropanol was added and incubated for 1 h at −20 degrees C (to precipitate nucleic acids). Tubes were then centrifuged for 20 min at 14000 r.p.m. (20,817g) at 4 degrees C and the supernatant removed. Pellets were washed with 70% ethanol and centrifuged at 14000 r.p.m. (20817g) for 5 min at 4 degrees C. After removing ethanol and air-drying, pellets were re-suspended in 0.2 ml of RNase free sterile water. DNA was removed using the Turbo DNA-free kit (Life Technologies), increasing the incubation time to 1 h to ensure rigorous DNA removal. After this step, samples were taken through the protocol supplied with the FastRNA Pro Soil-Direct kit to the end (starting at the RNA Matrix and RNA Slurry addition step), including the column purification step to remove residual humic acids (see FastRNA Pro Soil-Direct Kit manual). Extraction blanks were performed (adding sterile water instead of sample) to ensure that aerosolized contaminants did not enter sample and reagent tubes during the extraction process. Absence of DNA and RNA contamination was confirmed by no visible amplification of small subunit (SSU) ribosomal RNA (rRNA) and rRNA genes from extraction blanks after 35 cycles of PCR and RT–PCR.
After RNA extraction, the MEGA-Clear RNA Purification Kit (Life Technologies) was used to purify the RNA. This kit removes short RNA fragments (mostly produced during the extraction protocol) and residual inhibitors (that is, humics). We followed the protocol all the way through the optional precipitation/concentration step, re-suspending the RNA pellet in 10 ul of RNase-free sterile water. Before cDNA amplification, the removal of contaminating DNA in RNA extracts was confirmed by the absence of visible amplification of SSU rRNA genes after 35 cycles of PCR using the RNA extracts as template.
cDNA amplification and Illumina sequencing: Five microlitres of purified RNA was used as template for whole-cDNA amplification using the Ovation RNA-Seq v2 System (NuGEN technologies, http://www.nugeninc.com/nugen/index.cfm/products/cs/ngs/rna-seq-v2/). We followed the manufacturer’s instructions for cDNA amplification, and the resulting quantity of cDNA was checked on a Nanodrop (Thermo Scientific) and Fluorometer (Qubit 2.0, Life Technologies). Quality of the amplified cDNA was checked on a Bioanalyzer (Agilent Biotechnologies) before Illumina sequencing. Illumina library preparation and paired-end sequencing was performed at the University of Delaware Sequencing and Genotyping Center (Delaware Biotechnology Institute).
For more sampling information see www-odp.tamu.edu/publications/prelim/201_prel/201toc.html
The manuscript is at http://www.nature.com/nature/journal/v499/n7457/full/nature12230.html