GSM9288179: Δlsr2 mutant cells, replicate 3 (ChIP); Streptomyces venezuelae; ChIP-Seq
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.  RNA polymerase chromatin immunoprecipitation: Protein A-Sepharose beads were prepared in 1.5 mL tubes by resuspending 125 mg of the beads in 1.2 mL of Buffer A (0.02 M NaH2PO4, 150 mM NaCl, pH 8.0) and incubating at room temperature for 30 min. The beads were then aliquoted into separate 1.5 mL tubes for each sample (100 µL slurry per sample). The beads were equilibrated by removing the storage buffer and adding 1 mL of IP buffer with BSA (100 mM Tris pH 8.0, 300 mM NaCl, 2% Triton X-100, 0.1 mg/mL Bovine Serum Albumin). Beads were centrifuged at 3,000 rpm for 1 min at 4°C and allowed to settle on ice for 2 min. This wash step was repeated a second time, and the beads were resuspended in 100 µL IP buffer with BSA. To pre-clear the lysate, the remaining clarified lysate from after DNase treatment (~1 mL) was added to the equilibrated beads and incubated with gentle shaking at 4°C for 2 h. The beads were pelleted as above, the supernatant (pre-cleared lysate) was transferred to a clean tube, and 10 µL of anti-RNA polymerase antibody (developed in mouse) was added to each sample. The samples were incubated overnight with gentle shaking at 4°C. After removing the lysate, the beads were washed three times with 1 mL of Buffer A and stored at 4°C. The next day, the beads were equilibrated as before in IP buffer with BSA. The antibody-lysate mixture was added to the beads and incubated with gentle shaking at 4°C for 4 h. The beads were centrifuged as above and most of the supernatant discarded by removing with a pipette. The beads were washed at room temperature with 1 mL of each of the following solutions in order: ·       Wash buffer A (100 mM Tris, pH 8.0; 250 mM LiCl; 2% Triton X-100; 1 mM EDTA) ·       Wash buffer B (100 mM Tris, pH 8.0; 500 mM NaCl; 1% Triton X-100; 0.1% sodium deoxycholate; 1 mM EDTA) ·       Wash buffer C (10 mM Tris, pH 8.0; 500 mM NaCl; 1% Triton X-100; 1 mM EDTA) ·       IP buffer with EDTA (100 mM Tris, pH 8.0; 300 mM NaCl; 2% Triton X-100; 1 mM EDTA) ·       TE buffer (10 mM Tris, pH 8.0; 1 mM EDTA) After washing, the beads were resuspended in 500 µL elution buffer and protein-DNA crosslinking was reversed by incubating the samples at 65°C overnight. Samples were treated with RNase A and proteinase K, and the DNA precipitated. 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.