ena-STUDY-Department of Agricultural Biotechnology-19-12-2017-10:14:56:075-92
Transcriptome analysis of B. megaterium under acid stress
The bacterial culture was grown in culture media of pH4.5. Total RNA was isolated using Trizol method. The bacterial mRNA was enriched from total RNA by removing the 16s and 23s ribosomal RNAs through MICROBExpress kit (Ambion/Life tech). The mRNA was then reverse transcribed into first-strand cDNA using SuperScript II reverse transcriptase (Invitrogen) and random hexamer primer as per manufacturer's protocol. The single stranded cDNA was then converted to ds cDNA. The paired-end cDNA sequencing libraries were prepared using illumina TruSeq stranded mRNA Library Preparation Kit as per the described protocol. Briefly, ds cDNA samples were subjected to Covaris shearing followed by end-repair of overhangs resulting from shearing. The end–repaired fragments were A-tailed, adapter ligated and then enriched by limited no of PCR cycles. Library quantification and qualification was performed using DNA High Sensitivity Assay Kit.
The bacterial culture was grown in culture media of pH4.5. Total RNA was isolated using Trizol method. The bacterial mRNA was enriched from total RNA by removing the 16s and 23s ribosomal RNAs through MICROBExpress kit (Ambion/Life tech). The mRNA was then reverse transcribed into first-strand cDNA using SuperScript II reverse transcriptase (Invitrogen) and random hexamer primer as per manufacturer's protocol. The single stranded cDNA was then converted to ds cDNA. The paired-end cDNA sequencing libraries were prepared using illumina TruSeq stranded mRNA Library Preparation Kit as per the described protocol. Briefly, ds cDNA samples were subjected to Covaris shearing followed by end-repair of overhangs resulting from shearing. The end–repaired fragments were A-tailed, adapter ligated and then enriched by limited no of PCR cycles. Library quantification and qualification was performed using DNA High Sensitivity Assay Kit.