4 protocols
normalization data transformation protocol
The sample sources of individual reads were determined by the barcoding. The barcodes for the minus and plus TAP treatments were ACTTGA and CGATGT, respectively, for the E. coli RNA, and CAGATC and ATCACG, respectively, for the S. coelicolor RNA. RNA sequences from each of the two differential analyses were processed and mapped to the corresponding genomes as a service provided by vertis Biotechnologie AG, Germany (www.vertis-biotech.com). This involved trimming adaptor sequence and masking for low-quality sequence. For each of the 4 libraries (2 bacterial samples x 2 treatments), we counted the number of times each nucleotide position was the first in a sequence read using a simple script (unpubl. resource). The forward and reverse strands were then processed separately. Genome_build: The reference genomes used for E. coli K12 strain BW25113 and S. coelicolor A3(2) strain M145 were U00096.2 and AL645882, respectively. Supplementary_files_format_and_content: BedGraphs of the reads before and after TAP treatment for each of the samples. Separate files are provided for the forward and reverse strands.
nucleic acid library construction protocol
RNA was isolated from the pellet of E. coli cells using a well-established, published protocol (Kime et al. 2008. In RNA Turnover in Bacteria, Archaea and Organelles, Vol 447 (ed. LE Maquat, CM Arraiano), pp. 215). The cell pellet of S. coelicolor was resuspended in Kirby mix (Kieser et al. 2000. Practical Streptomyces Genetics. The John Innes Foundation, Norwich.), 100 µL per 1 O.D.600nm unit, and transferred to Lysing Matrix B tubes containing fine silica beads (MP Biomedical). Tubes were then placed in a high-speed benchtop homogenizer (Fastprep-24, MP Biomedical; set at 6.5 M/s). Cells were lysed by three cycles of homogenisation for 1 min with cooling between each cycle in an ice-water bath for 1 min. The lysates were extracted using an equal volume of acidic phenol: chloroform: isoamyl alcohol (50: 50: 1) and then chloroform: isoamyl alcohol (49: 1). Nucleic acid in the aqueous phase was precipitated by adding NaCl to 150 mM and 2.5 x volumes of 100% [v/v] ethanol, chilling at -20°C for 1 h, and then harvested by centrifugation (13,000 x g for 30 min at 4°C) . The nucleic acid pellet was washed twice with 700 µL of 70% [v/v] ethanol, air dried for 5 min and resuspended in RNase-free water. Contaminating DNA was removed from both E. coli and S. coelicolor by incubating with DNase as described by the vendor (Ambion) and extracted with phenol: chloroform as described above. The concentration and integrity of RNA samples were determined using a NanoDropTM 1000 spectrophotometer (Thermo Fisher Scientific) and agarose gel electrophoresis (Kime et al. 2008), respectively. Samples were enriched for mRNA using MICROBExpress-Bacteria beads, as described by the manufacturer (Ambion). Libraries were constructed by vertis Biotechnologie AG, Germany (www.vertis-biotech.com) as a service that included treating an aliquot of each RNA sample with TAP. The 5'-sequencing adaptor was ligated to transcripts prior to fragmentation, thereby allowing the 5' ends of both long and short transcripts to be detected. Each of the 4 libraries was constructed using a different barcode.
growth protocol
Escherichia coli K12 strain BW25113 (Datsenko & Wanner 2000. PNAS 97: 6640) was cultivated in 100 mL of LB broth ( Miller 1972. In: Experiments in molecular genetics. Cold Spring Harbor Laboratory, NY) in a 250 mL Erlenmeyer flask at 37°C with shaking (250 rpm) to an O.D.600nm of 0.6. S. coelicolor A3(2) strain M145 (Kieser et al. 2000. Practical Streptomyces Genetics. The John Innes Foundation, Norwich.) was incubated in YEME broth (Kieser et al. 2000) at 30°C with shaking until the mycelia became pigmented.
sample treatment protocol
At the appropriate phase of growth, a one-eighth volume of stop solution (95% [v/v] ethanol; 5% [v/v] phenol) was added to a culture to inhibit cell metabolism (Kime et al. 2008. In RNA Turnover in Bacteria, Archaea and Organelles, Vol 447 (ed. LE Maquat, CM Arraiano), pp. 215), and the cells were harvested by centrifugation. When necessary, cell pellets were stored frozen at -80C.