GSM2775065: M_rep2; Methanocella conradii; RNA-Seq
Total RNA were extracted using AllPrep DNA/RNA Mini Kit (Qiagen, Germany) according to the manufacture's instruction. RLT plus buffer was supplemented with 2% v/v 2 M DTT (Sangon Biotech, China). 600 µl RLT Plus buffer was added to each cell pellet. Cell pellets of P and MP were homogenized for 120 s in total (5.0 m/s, 3×40 s with 2 min intervals on ice) in a FastPrep-24 (MP Biomedicals, USA) with 0.5 g RNase free beating beads (0.1 mm, Sigma, USA). Cell pellets of M were homogenized at 5.0 m/s for 20 s without beating beads. Residue DNA digestion, verification of DNA absence and RNA purification were carried out as previously described (Liu et al., 2014, Applied and environmental microbiology 80:4668-4676;). RNA was finally dissolved in 75 µl RNase free H2O. RNA integrity values (RIN) were determined by an Agilent 2100 Bioanalyzer (Agilent Technologies, USA) according to the manufacturer's instruction mRNA enrichment was carried out by using the Ribo-Zero™ Magnetic Kit for Bacteria (Epicenter, USA) and RNA libraries were prepared for strand specific sequencing using protocols as previously described (Parkhomchuk et al., 2009, Nucleic Acids Res, 37:e123)