GSM5172039: FeS2 rep C; Methanosarcina barkeri; RNA-Seq
To harvest biomass, M. barkeri MS cells were subjected to vacuum filtration at low pressure (5 psi) onto a 47 mm 0.2-µm Supor 200 PES filter (Pall, Port Washington, NY) within an anaerobic chamber. Using flame-sterilized scissors, each filter was cut in half and each half transferred to a 1.5 mL cryotube before being removed from the anaerobic chamber and immediately flash frozen in liquid nitrogen. Frozen cells were then stored at -80 °C until processing. Total RNA from M. barkeri was extracted using TRIzol reagent (Invitrogen) following the manufacturer's protocol with slight modification. Individual frozen half filters containing M. barkeri cells were transferred to a Lysis E tube (MP Biomedicals) on ice and 1 mL of TRIzol was immediately added to the frozen filter. TRIzol-treated M. barkeri cells were subjected to three cycles of 40 seconds of bead beating followed by resting for five minutes at room temperature (~20 °C). Two hundred µL of molecular grade chloroform was added to each tube and each tube was inverted to mix, incubated at room temperature for three minutes, and centrifuged for 15 minutes at 12,000 x g at 4 °C. The upper aqueous phase containing RNA was carefully transferred to a clean 2 mL tube. To precipitate RNA, 0.5 mL of pre-chilled (4 °C) 100% molecular grade isopropanol was added to each tube and tubes and their contents were incubated on ice for 10 minutes followed by centrifugation for 10 minutes at 12,000 x g at 4 °C. The supernatant was discarded, and 1 mL of 75% molecular grade ethanol was added to wash the RNA. After centrifugation for 5 minutes at 7,500 x g at 4 °C, the supernatant was removed, and the RNA pellet was air dried for 5-10 minutes. Total RNA was dissolved in 50 µL RNA-grade water (Fisher Scientific, Waltham, MA) by incubating in a 55 °C heat block for 10 minutes. RNA was treated with Turbo DNase (Invitrogen) to remove residual DNA per manufacturer's instructions. After DNase treatment, the remaining total RNA was subjected to a second round of isopropanol precipitation, ethanol wash, and resuspension 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. barkeri MS-specific oligos designed using the sequences for M. barkeri MS'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).