GSM9288149: Δlsr2ΔlsrL mutant cells, replicate 2 (IPOD-HR); Streptomyces venezuelae; OTHER
Lysis of S. venezuelae cell pellets: Each of the washed cell pellets from above were resuspended in 1.5 mL lysis buffer (10 mM Tris-HCl pH 8.0, 50 mM NaCl, 14 mg/ml lysozyme, 0.8% Triton X-100, 1× protease inhibitor) and incubated at 37°C for 30 min. The samples were incubated on ice for 2 min, then sonicated for six cycles of 15 s continuous sonication followed by 45 s on ice (output 40%, 4). Cell debris was removed by centrifugation for 20 min at 13,000 rpm at 4°C and the supernatant was transferred to a 15 mL Falcon tube (~1.8 mL).  Determination of optimal DNase treatment conditions: One of the six replicates was used to determine the appropriate conditions for DNase treatment that would yield DNA fragments between 100-200 bp, as we found that required treatment was different for each sample. To determine the appropriate time for DNase treatment, 200 µL of 10× Turbo DNase Buffer, 10 µL Turbo DNase, and RNase A to a final concentration of 15 µg/mL were added to this sample, and incubated for 90 min at 37°C. Every 10 min during the 90-min time course, a 50 µL aliquot was collected from each sample, mixed with 10 µL of 0.5 M EDTA (pH 8), and incubated on ice to inhibit further DNase digestion. Once all aliquots had been collected, they were subjected to phenol-chloroform extraction, and 5-10 µL of the upper phase was run on a 2% agarose gel. The time that yielded the most DNA between 100-200 bp was used to digest the remaining five replicates.  DNase treatment: The remaining five replicates were DNase-treated using the experimentally determined optimal conditions. After DNase digestion, 500 µL of 0.5 M EDTA (pH 8.0) was added to quench the reaction. The samples were clarified by centrifuging at 7,500 rpm for 10 min at 4°C. A 50 µL aliquot (IPOD input DNA) was removed and diluted 1:9 in elution buffer (50 mM Tris, pH 8.0; 10 mM EDTA; 1% SDS) and kept on ice until the reverse crosslinking step. A 100 µL aliquot of the clarified lysate was phenol-chloroform extracted and 5 µL of the upper (DNA) phase was run on a 2% agarose gel to confirm that the DNase treatment was successful and had yielded DNA with a maxima centered between 100-200 bp.  IPOD-HR: One milliliter of the resulting clarified lysate was transferred to a 15 mL Falcon tube and mixed with an equal volume of 100 mM Tris base. An equal volume of 25:24:1 phenol:chloroform:isoamyl alcohol was added, the sample vortexed, and incubated for 10 min at room temperature. After incubation, the sample was centrifuged at 7,500 rpm for 5 min at 4°C, allowing the formation of a white disc at the aqueous–organic interface enriched for protein–DNA complexes. Both the upper and lower aqueous phases were removed with a pipette and discarded. The remaining interface disc was washed with 350 µL TE buffer (10 mM Tris, pH 8.0; 1 mM EDTA), 350 µL 100 mM Tris base, and 700 µL 24:1 chloroform:isoamyl alcohol. The sample was vortexed and centrifuged as above. The upper and lower aqueous phases were removed again, and the interface disc was washed with 1 mL TE buffer and 1 mL 24:1 chloroform:isoamyl alcohol. The sample was vortexed and centrifuged as above, and both upper and lower aqueous phases were discarded. The interface disc was resuspended in 700 µL of elution buffer by vortexing. To reverse protein-DNA crosslinking, the input and IPOD-HR samples were incubated at 65°C overnight. The next day, the samples were allowed to cool, and RNase A was added to each input and IPOD-HR sample to a final concentration of 60 µg/mL. The samples were incubated at 37°C for 2 h. Proteinase K was then added to a final concentration of 120 µg/mL and incubated at 50°C for 2 h. DNA was phenol-chloroform extracted, the upper phase was transferred to a clean tube and precipitated by the addition of 2 volumes of cold 95% ethanol and 0.1 volume of precipitation buffer (5 M CH3COONa/CH3COOH, pH 5.2) and incubation at -20°C overnight. The next day, samples were centrifuged at 13,000 rpm for 30 min at 4°C, and the supernatant discarded. The DNA pellet was washed with ice-cold 70% ethanol and dried by placing the tube up-side-down on a paper towel. The DNA pellet was resuspended in 30 µL 10 mM Tris-HCl, pH 8.0. All purified DNA samples were run on an Agilent TapeStation to assess DNA quality prior to library preparation. Libraries were prepared for Illumina sequencing using the NEBNext Ultra II DNA kit (New England Biolabs). The samples were pooled into libraries and sequenced on an Illumina NextSeq 2000, in a P3, 2x50bp configuration at the McMaster Genomics Facility.