GSM7506310: Tyg bile salts, replicate 3; Bacteroides thetaiotaomicron; OTHER
After end-repair, TruSeq sequencing adapters were ligated to the DNA fragments. Finally, the DNA was PCR-amplified to about 10-20 ng/μl using a high fidelity DNA polymerase. The PCR amplified samples were purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and were analyzed by capillary electrophoresis We incubated the culture at 37°C until it reached the indicated OD, at which point we took a 2 mL aliquot, pelleted it, discarded the supernatant, and stored the bacterial samples at -20°C until further processing.  Bacterial pellets were resuspended in 100 μL of PBS containing 250 μg lysozyme, mixed with 100 μL lysis buffer (100 mM Tris-HCl pH 8.5, 200 mM NaCl, 0.2% SDS, 5 mM EDTA), and lysed by incu-bating at 37°C for 5 min. Proteins were then degraded by addition of 1 μL of Proteinase K (NEB #P8107) and incubation at 56°C for 30 min, following which RNA was digested with 0.5 μL of RNase A (Thermo #EN0531) and incubation at 56°C for 5 min. The remaining gDNA was purified from each sample via phenol-chloroform extraction, followed by ethanol precipitation.  The genomic regions coding for gRNAs and arrays were PCR-amplified in a 50 µL reaction con-taining 100 ng gDNA with oligonucleotides AWO-577/AWO-578 (annealing temperature: 55°C) and AWO-575/AWO-576 (annealing temperature: 63°C).