title: GSM3331439: RNA A2; Clostridium drakei; RNA-Seq
library_construction_protocol: The cells were resuspended in 500 µL lysis buffer composed of 20 mM Tris-HCl (pH 7.4), 140 mM NaCl, 5 mM MgCl2, and 1% Triton X-100. Resuspended cells were frozen in liquid nitrogen and then ground with pestle and mortar. The powdered cells were thawed and the cell debris was removed by centrifugation at 4000 g for 15 min at 4 °C. Total RNA was then isolated using TRIzol (Thermo Scientific). To remove residual genomic DNA in the RNA sample, 4 U of rDNase I (Ambion) was applied to the isolated RNA for 1 h at 37 °C, followed by incubation at 75 °C for 10 min. Ribosomal RNAs (rRNAs) were removed by using the Ribo-ZeroTM rRNA Removal Kit for Meta-bacteria (Epicentre), and the rRNA-depleted RNA quality was checked using the Agilent 2200 TapeStation system (Agilent Technologies). Using the TruSeq Stranded mRNA Library Prep Kit (Illumina), the rRNA-depleted RNAs were converted into RNA-Seq libraries, which were then sequenced by the 150 bp read recipe with an Illumina MiseqTM system. The cells were resuspended in 500 µL lysis buffer composed of 20 mM Tris-HCl (pH 7.4), 140 mM NaCl, 5 mM MgCl2, and 1% Triton X-100. Resuspended cells were frozen in liquid nitrogen and then ground with pestle and mortar. The powdered cells were thawed and the cell debris was removed by centrifugation at 4000 g for 15 min at 4 °C. Total RNA was then isolated using TRIzol (Thermo Scientific). To remove residual genomic DNA in the RNA sample, 4 U of rDNase I (Ambion) was applied to the isolated RNA for 1 h at 37 °C, followed by incubation at 75 °C for 10 min. Ribosomal RNAs (rRNAs) were removed by using the Ribo-ZeroTM rRNA Removal Kit for Meta-bacteria (Epicentre), and the rRNA-depleted RNA quality was checked using the Agilent 2200 TapeStation system (Agilent Technologies). Using the TruSeq Stranded mRNA Library Prep Kit (Illumina), the rRNA-depleted RNAs were converted into RNA-Seq libraries, which were then sequenced by the 150 bp read recipe with an Illumina MiseqTM system.