GSM1571887: MGA3_WT; Bacillus methanolicus; RNA-Seq
For the extraction of total RNA the NucleoSpin RNA isolation kit (MACHEREY-NAGEL, Düren, Germany) and the RNase-free DNase set (QIAGEN, Hilden, Germany) were used according to the manufacturer’s instructions. The total RNA was isolated individually for each cultivation condition. The quality and quantity of the isolated RNA was assessed by PCR with gene-specific primers (Supplementary Table 1) and with capillary gel electrophoresis (Agilent Bioanalyzer 2100 system using the Agilent RNA 6000 Pico kit; Agilent Technologies, Böblingen, Germany). The extracted RNA samples were pooled in equal parts and the pool of total RNA was subsequently used for the preparation of two different cDNA libraries. The cDNA libraries of B. methanolicus MGA3 were prepared according to two different protocols. One approach focused on the enrichment of 5’‑ends of primary transcripts, while the other method allowed the analysis of the whole transcriptome. Both cDNA library preparation protocols have been previously described in detail  (Pfeifer-Sancar et al. 2013, BMC Genomics). In the present study, the experimental workflow was changed at two steps. The first difference is that the amplification of cDNA fragments was customized by adding the PCR additive betaine to a final concentration of 2.7 M in each PCR reaction. Secondly, after cDNA amplification the two libraries were purified and size-selected via gel electrophoresis for fragment sizes between 100 and 800 bp.
GEO
GSM1571887