GSM6697326: FeS2-grown NH3-amended cells, biol rep 2; Methanococcus maripaludis; RNA-Seq
To harvest biomass, M. mari S2 cells were harvested via cetnrifugation under an anoxic headspace. Supernatant was decanted and the cell pellets were flash frozen in liquid nitrogen. Frozen cells were then stored at -80 °C until processing. Total RNA from M. maripaludis was extracted using TRIzol reagent (Invitrogen) following the manufacturer's protocol with slight modification. One mL of TRIzol was added to cell pellets and the resuspended cells were transferred to Lysis E tubes (MP Biomedicals, Irvine, CA) on ice. The cell samples were mechanically lysed by three cycles of 40 sec of bead beating and five mins of rest at room temperature (~20 °C). After the lysis procedure, 200 µL of molecular grade chloroform was added, the tubes were mixed by inversion and allowed to incubate for three mins at room temperature. The tubes were then centrifuged for 15 mins at 12,000 x g at 4°C and the upper aqueous phase containing RNA was transferred by pipette into a clean two mL tube. RNA was precipitated by the addition of 0.5 mL of 100% molecular grade isopropanol that had been pre-chilled to 4°C followed by ten mins incubation on ice. RNA was then pelleted by centrifugation for ten mins at 12,000 x g at 4°C. The supernatant was removed by pipette, and the RNA was washed in one mL of 75 % molecular grade ethanol. RNA was pelleted by centrifugation for five mins at 7,500 x g at 4 °C, the supernatant was removed, and the RNA pellet was air dried for ten mins. Once dried, the RNA was resuspended in 50 µL of RNA-free H2O (Fisher Scientific, Waltham, MA) by incubating at 55°C in a heat block for ten mins. RNA was treated to remove residual DNA by the addition of Turbo DNase (Invitrogen, Waltham, MA) according to the manufacturer's instructions. The RNA was then subjected to a second round of precipitation, washing, drying, and resuspension steps as described above. Quality control was performed with a Bioanalyzer (Agilent, CA, USA), and rRNA reduction was performed with a RiboZero-Bacteria rRNA removal kit (Illumina, CA, USA) with the addition of custom M. maripaludis S2-specific oligos designed using the sequences for M. maripaludis S2's large and small ribosomal subunits. Stranded cDNA libraries were prepared from rRNA-depleted mRNA using the TruSeq Stranded Total and mRNA kit (Illumina, CA, USA).