Pfeifer-Sancar, Katharina; Mentz, Almut; Rückert, Christian ; Kalinowski, Jörn: Comprehensive analysis of the Corynebacterium glutamicum transcriptome using an improved RNAseq technique. In: BMC Genomics. Jg.14 H. 1. 2013
Inhalt
- Results
- Development of native 5′-end and whole transcript RNAseq protocols
- Data generation by Illumina sequencing and mapping of DNA sequence reads to the Corynebacterium glutamicum ATCC13032 genome
- Comprehensive identification of transcription start sites from the native 5′-end data set
- Identification of σA-dependent promoters
- Re-annotation of coding sequences
- Characteristics of 5′-UTRs and leaderless mRNAs
- Analysis of ribosome binding sites
- Identification of operon structures based on the whole transcriptome data set
- Description and classification of novel transcripts
- Transcript ends and predicted rho-independent terminators
- Discussion
- σA-dependent promoters in C. glutamicum
- Characterization of 5′-UTRs indicate novel regulatory elements
- Abundance of leaderless mRNAs in C. glutamicum
- RNAseq data reveal AGGag as ribosome binding site for C. glutamicum
- Revealing operon structures for C. glutamicum
- Detection of novel transcripts unveil a huge amount of antisense transcription
- Transcript ends and predicted rho-independent terminators
- Conclusions
- Methods
- Bacterial strains, oligonucleotide and culture media
- Growth conditions for cell sampling
- RNA isolation procedures
- The native 5′-end protocol
- The whole transcriptome protocol
- Depletion of ribosomal RNA
- Phenol-Chloroform Isoamyl alcohol extraction
- RNA fragmentation
- Terminator 5′-phosphate-dependent exonuclease treatment
- 5′-end repair
- RNA adapter ligation
- Preparation of the stem-loop DNA adapter
- Reverse transcription and tagging of the 3′-end of the cDNA in a single step
- Amplification of the cDNA and purification of cDNA library
- Sequencing of the cDNA library
- Bioinformatics data analysis
- Additional files
- Abbreviations
- Competing interests
- Authors’ contributions
- Acknowledgements
- References