BioSample

Protocols: Streptomyces albus S4 strains were grown in Lennox Broth (Kieser et al. Practical Streptomyces Genetics. The John Innes Foundation. Norwich, United Kingdom) whilst shaking for 48 hours at 28 degrees Celsisus. The same as P-MTAB-39829 exception that an an Active Motif EpiShear sonicatior (30% amplitude, 30s on, 30s off for a total time of 13 minutes) was used to shear DNA. DNA libraries were made using the NEBNext Ultra DNA Library Prep Method. In brief, samples were initially quantified using High Sensitivity dsDNA Qubit assay (Thermo Q32854). Ten nanograms of each DNA sample was then sheared via sonication on the Covaris S2 system to an average size of ~200 bp. This was confirmed by checking samples on Agilent High Sensitivity D1000 Screentape. Samples were then end repaired by addition of a single A nucleotide added to the 3’ end of the DNA fragments and (10-fold diluted) adaptors ligated according to NEB’s instructions. Due to the small amount of starting material, no size selection was performed, yet the adaptor ligated fragments were bead purified according to NEB’s instructions, substituting Ampure XP beads for AxyPrep PCR clean up beads (Axygen). The purified adaptor ligated DNA fragments were then PCR amplified according to NEB’s instructions, incorporating a different 3’ 6 bp index sequence into each sample, which would allow post-sequencing demultiplexing. The PCR products were then bead purified according to NEB’s instructions, substituting Ampure XP beads for AxyPrep PCR clean up beads (Axygen). The final libraries were assessed on Agilent High Sensitivity D1000 Screentape as above, before being quantified by picogreen (Thermo Q33130). One hundred nanograms of each DNA library was then added to a pool for subsequent sequencing.