GSM6735434: Z-245, H2/CO2, biol rep 2; Methanothermobacter thermautotrophicus; RNA-Seq
A hot phenol method was used for RNA extraction. The samples were thawed on ice and centrifuged at 4°C and 4100 × g for 10 min, the supernatant discarded, and the samples resuspended in 800 µL of RNase free water. The 800 µL were mixed with 950 µL of saturated phenol (Sigma-Aldrich, Merck, Germany) and 115 μL of a lysis solution in Lysing Matrix B (MP Biomedicals Germany GmbH, Eschwege, Germany). The lysis solution contained sodium acetate (20 mM pH 5.2), sodium dodecyl sulfate (SDS; 0.5 % v/v), ethylenediaminetetraacetic acid (EDTA; 1 mM), and DNase/RNase-free distilled water (DI; Invitrogen, Thermo Fischer Scientific, Germany). The cells were homogenized for one cycle (5 x 40 s at 6 m/s and 20 s off) in a FastPrep-24TM 5G bead-beater (MP Biomedicals Germany GmbH, Eschwege, Germany) and then centrifuged at room temperature and 21130 × g for 10 min (5424 Eppendorf, Hamburg, Germany). The top layer was placed into 600 µL of phenol-chloroform-isoamyl alcohol (ROTI® Aqua-P/C/I, Carl Roth, Karlsruhe, Germany) and centrifuged as in the previous step. This step was repeated twice. The top layer was added to 1.2 mL of ethanol (undenatured absolute, SERVA Electrophoresis GmbH, Heidelberg, Germany) and left overnight at -80 °C to precipitate the RNA. The following day, the tubes were centrifuged at 4°C and 21130 × g for 10 min, and then the supernatant was removed. The RNA pellet was washed with 1 mL of 75% v/v ethanol solution and centrifuged as in the previous step. The supernatant was pipetted off, and the pellet was resuspended with 53 µL of DNase/RNase-free distilled water. The RNA was cleaned (which included DNA depletion) and concentrated using the RNA Clean & Concentrator kit (Zymo Research, Irvine, CA, United States), according to the manufacturer's instructions. The cleaning and concentrating protocols were repeated four times. RNA libraries for RNA-seq were prepared using 100 ng of RNA and the TruSeq™ Stranded Total RNA Kit with Ribo-Zero™ Plus following manufacturer's protocols.