Whole genome sequencing of spontaneous resistant mutants of Mycobacterium tuberculosis H37Rv selectec in the presence of BVL3572S and amino acids
Whole genome sequencing of spontaneous resistant mutants of Mycobacterium tuberculosis H37Rv selectec in the presence of BVL3572S and amino acids
7 mL of exponentially growing cultures were harvested by centrifugation at 3,500 x g for 5 min. Plates containing Middlebrook 7H10 or 7H9 solid medium supplemented with glycerol 0.2%, Tween 80 0.05% and OADC 10% were prepared with BVL3572S alone (7H9) or in the presence of L-His 3 mM (7H9) or L-Ala 3 mM (7H10). 7H9 solid medium was used for mutant selection with L-His or alone because we suspected our 7H10 stock to contain trace amounts of L-Ala. Plates were inoculated with 106, 107, or 108 CFUs of a weeks-old culture of Mtb H37Rv (OD600 nm > 1) and incubated at 37 °C for at least 4 weeks. Individual colonies were picked and cultured until exponential phase in the presence of the corresponding amino acid if applicable.  Pellets were resuspended in 500 µL of UltraPure™ Phenol:Chloroform:Isoamyl Alcohol (Thermo Fisher 15593031), and homogenized in impact-resistant 2 mL tubes containing 0.1 mm silica spheres (Lysing Matrix B, MP biomedicals) using a FastPrep FP120 cell disrupter (Thermo Fisher Scientific) at 6.0 Hz for 40 s. Samples were mixed with 500 µL TE buffer (10 mM Tris pH 8.5, 1 mM EDTA) and incubated for 5 min at room temperature before centrifugation (10 min, 10,000 rpm, 4 °C). The aqueous phase (400 µL) was transferred to a new tube, to which 80 µL sodium acetate (4 M, pH 5.5) and 800 µL of 100% cold ethanol were added. DNA was precipitated overnight at - 20 °C. After centrifugation (30 min, 12,000 rpm, 4 °C), the supernatant was removed, and the pellet was washed twice with 700 µL of 70% cold ethanol followed by centrifugation (15 min, 12,000 rpm, 4 °C). Pellets were air-dried for around 30 min and resuspended in 35 µL nuclease-free water. DNA was quantified by Qubit dsDNA BR assay kit (ThermoFisher Scientific). The genomic DNA was randomly sheared into short fragments. The obtained fragments were end repaired, A-tailed and further ligated with Illumina adapter. The resulting fragments with adapters were size-selected and PCR-amplified before proceeding for purification. The library was quantified through Qubit and qPCR, and size distribution detected with fragment analyzer. Library preparation kit: Novogene NGS DNA Library Prep Set (Cat No.PT004) 