Dataset: Whole community metatranscriptomes
Deployment: JRES-201

RNA was extracted from 20 g of sediment from the Peru Margin and 16 g of the Canterbury Basin core samples using the RNA PowerSoil Total RNA Isolation Kit (MoBIO Laboratories, Carlsbad, CA) following a modified manufacturer’s protocol. Modifications included: ten cycles of homogenization for 1-minute intervals with 1 minute rest between intervals using a FastPrep benchtop homogenizer (MP Biomedicals, Santa Ana, CA) set to 4.0 m/s and increasing the incubation time to 45 min following the addition of SR4 buffer. Trace DNA was removed by treatment with Turbo DNA-free (Life Technologies, Grand Island, NY) for 60 minutes at 37 degrees C. A final RNA purification step was performed using the MEGAclear kit (Life Technologies, USA). In order to avoid contamination, all manipulations were carried out in a dedicated PCR hood (AirClean Systems, USA) for RNA work. An extraction blank was also carried through the entire procedure to control for kit contamination and served as "negative control". Removal of carry-over DNA in RNA extracts was confirmed by the absence of visible amplification of the V4 hypervariable region of SSU rDNA after 35 cycles of PCR using the RNA extracts as template with key-tagged bacterial primers (Flores et al., 2011). PCR conditions were: 95 degrees C for 2 minutes followed by 35 cycles of 95 degrees C for 15 seconds, 53 degrees C for 45 seconds and 68 degrees C for 45 seconds with a final incubation of 68 degrees C for 3 minutes. Total RNA was used as template for cDNA amplification using the Ovation RNA-Seq System V2 kit (NuGEN, San Carlos, CA) that uses random hexamers to initiate reverse transcription. Purification of double stranded cDNA was performed with Agencourt RNAClean XP Purification Beads following the instructions provided by NuGEN. Two replicate extractions and reverse transcriptions were performed per sample. The quantity of amplified cDNA was evaluated using a fluorometer (Qubit 2.0, Life Technologies).