Dataset: poly-A mRNA
Deployment: JRES-317

During IODP leg 317 expedition (JOIDES Resolution), a sediment core was drilled at Site U1352 in 344 m water depth. A total depth of 1927.5 mbsf was cored, spanning the Holocene to late Eocene periods. For this work, core sections were obtained from this site from 3.76 mbsf, 11.60 mbsf, 24.60 mbsf, 345.50 mbsf, and 403.10 mbsf.

RNA was extracted from 16 g of sediment from the Canterbury 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 3’-DGE System (NuGEN, San Carlos, CA) to obtain an enriched eukaryotic metatranscriptome through selection of polyA transcripts. Double-stranded cDNA was purified using the DNA Clean & Concentrator kit (Zymo Research) as described in the modified user guide (https://www.biolynx.ca/pdf/admin/nu_threedgeuserguide.pdf) provided by NuGEN. The quantity of amplified cDNA was evaluated using a fluorometer (Qubit 2.0, Life Technologies).