GSM4818214: MsmegWT5minR1; Mycolicibacterium smegmatis; OTHER
We apply XR-seq in Mycolicibacterium smegmatis MC2 155, the cells were were grown with shaking at 37 °C in Luria Broth containing 0.5% (w/vol) Tween 80. Exponentially growing cells were transferred to R150 tissue culture dishes in volumes of 15 mL and were irradiated at an OD600 of ∼0.6 at room temperature with 100 J/m2. dishes were then incubated for 10 min at room temperature, and then cells were harvested, chilled, and maintained on ice. Excision products were then isolated using a modified Hirt procedure that employs shearing to assist cell lysis.  Cells were pelleted at 4 °C, resuspended in ice-cold TE [Tris (10 mM)-EDTA (1 mM) (pH 8.0)], transferred to ice cold Eppendorf tubes, and pelleted at 4 °C, and the supernatants were removed. Pellets were then resuspended with 360 μL of ice-cold TE. Then, 50 μL of room temperature 10% (wt/vol) SDS was added, and cells were lysed by passage through a 26G needle 40 times followed by incubation at room temperature for 20 min. A 120-μL volume of room temperature NaCl (5 M) was then added, tubes were gently mixed, and suspensions were incubated at 4 °C overnight. After centrifuging at high speed for 1 h in a microfuge at 4 °C, supernatants (about 480 μL each) were taken, and each was mixed with and incubated with 12 μL of RNaseA (R4642; Sigma) for 1 h at 37 °C, and then with 12 μL of proteinase K (P8107S; NEB) for 1.5 h at 60 °C. Samples were then extracted twice with phenol/chloroform/isoamyl alcohol and precipitated with ethanol. Samples were immunoprecipitated with an anti-CPD antibody and then ligated to adapters and processed for sequencing (XR-seq). XR-seq libraries were pooled and then sequenced on the HiSeq 2500Rapid Run platform at the UNC-Chapel Hill High-Throughput Sequencing Facility.