NextSeq 500 sequencing; Drug-specific signatures of the glycopeptide antibiotics dalbavancin, vancomycin and chlorobiphenyl-vancomycin in Streptomyces coelicolor
Drug-specific signatures of the glycopeptide antibiotics dalbavancin, vancomycin and chlorobiphenyl-vancomycin in Streptomyces coelicolor
Bacterial cultures Spores of Streptomyces coelicolor M600 were germinated and grown to mid-log phase (OD450 nm approx. 0.5) in liquid NMMP medium as described previously (Tran et al. 2019; Hong et al. 2005; Hong et al. 2004). Biological duplicate cultures were treated at this point by adding vancomycin (Sigma), dalbavancin or chlorobiphenyl-vancomycin to a final concentration of 10 ug/ml. A vehicle-only control (ethanol) in which cultures received no antibiotic treatment was also applied. Cultures were incubated for a further 30 min before harvesting for total RNA extraction.   Total RNA was isolated from mycelium harvested from 5 ml liquid cultures using an RNeasy Midi Kit (Qiagen) according to the manufacturer's instruction with some modifications. The cell pellet was resuspended in TE buffer containing lysozyme (10 mM Tris, pH 8, 1mM EDTA, 15 mg/ml lysozyme) and incubated at room temperature for 60 minutes. RLT buffer (Qiagen) was added (4 ml) and samples were sonicated 3 cycles ON-OFF on ice at 18 micron amplitude and for 20 seconds. Samples were then extracted twice with Phenol:Chloroform:Isoamyl Alcohol 25:24:1 saturated with 10 mM Tris, pH 8.0, 1 mM EDTA (2 ml) and once with chloroform (4 ml). Extracts were mixed with 100% ethanol and applied to RNeasy Midi columns. Purified RNA was eluted with 300 µl RNasefree water.  rRNA depletion (bacteria) pto  ribosomal RNA depletion protocol. Use the Illumina kit cat no MRZ11124C. Procedure: Add 2ug of total RNA in a total volume of 40ul with 4 ul of buffer and 8 ul of rRNA removal solution.Incubate at 68C for 10 min in the ThermomixerC . Brief spin. Incubate at room temperature for 5 min. Add 40ul of reaction mix to the washed beads. Immediately mix by pipetting up and down 10 times and vortex 10 sec at medium setting. Incubate 5 min at room temperature. Incubate 5 min at 50C. Place tube on the magnetic stand and incubate at least 1 min. Transfer the supernatant to a fresh tube. Measure the volume and add RNase free water to make the volume to 180ul. Precipitate the RNA by adding 18ul of 3 M NaOAC pH 5.5, 2 ul of glycogen, mix by vortex and add 3V of 100% ice cold ethanol. Incubate at -80C for 30 min, spin down in a refrigerated centrifuge for 30 min at 15.000 rpm. Remove all supernatant. Wash pellets in 70% ethanol by adding 500ul of ethanol and spinning for 5 min at 4C at 13000 rpm. Remove all supernatant and air dry the pellets. Resuspend the RNA in 7 ul of RNase free water. Quantify the RNA using Qubit Fluorometer (RNA broad range) and check the RNA using the TapeStation 4200 (Agilent Technologies) and RNA screen tape  (Agilent Technologies cat.5067 5576) to verify the success of the rRNA depletion. Library construction protocol. 100 ng of rRNA depleted RNA was used as input for library construction . The NebNext Ultra II Directional RNA library prep kit for Illumina (NEB E7760S) was used for library construction following the manufacturer's recommendations. Briefly the RNA is first fragmented at 94C for 15 minutes and then converted to cDNA . The cDNA is converted to double strand DNA and purified using SPRI select beads (Beckman Coulter cat B23318). The purified DNA is subjected to end prep to repair the ends before adaptor ligation ( n.b. the adaptor is diluted 25 fold before adding it to the reaction). The adaptor ligated DNA is purified using SPRI select beads and the DNA is amplified using the universal PCR primer and a library specific primer from the NEBNext Multiplex oligos for Illumina (set 1 NEB cat. E7335) for 11 PCR cycles. The DNA is purified using SPRI select beads and checked on the Tape Station 4200  (Agilent Technologies High sensitivity D1000 Screen tape cat 5067-5584) to determine the quality of the library and the size distribution of the DNA fragments. The libraries are quantified using the Qubit Fluorometer 2.0 using the DNA high sensitivity kit (Thermo Fisher ). Following quantification the libraries are normalized and sequenced. Libraries normalization: each library is diluted to a final concentration of 10nM using Illumina RSB buffer and further diluted  to 2 nM concentration. Equal volumes of each library are pooled in one tube and concentration is checked using NebNext Quant-IT library quantification kit for Illumina (NEB cat E 7630L). Final concentration if needed is adjusted to 2 nM according to the QPCR results.