title: GSM5471045: Ex1-Starved-5mL; Mycobacterium tuberculosis; OTHER
library_construction_protocol: After harvesting the cells, DNA was extracted using a published CTAB protocol (M. H. Larsen, K. Biermann, S. Tandberg, T. Hsu, W. R. Jacobs Jr, Genetic manipulation of Mycobacterium tuberculosis. Current protocols in microbiology 6, 10A. 12.11-10A. 12.21 (2007).) DNA was sheared using a focused-ultrasonicator (Covaris) and purified using a QIAquick PCR purification kit (Qiagen). Sheared DNA underwent end repair and A-tailing using NEBNext Ultra End Repair/dA-Tailing Module (New England Biolabs). Products were again purified with the QIAquick.  PCR purification kit (Qiagen), and then underwent adaptor ligation with T4 ligase (NEB) overnight. DNA of >100 base pairs was subsequently purified with AMPure XP beads (Beckman Coulter) and a DynaMag-2 magnet (Thermo Fisher). DNA products were PCR amplified using Choice Taq polymerase (Denville Scientific), and purified with QIAquick PCR purification kit (Qiagen). Appropriate DNA size (300-400 BP) was selected using a Pippin Prep machine (Sage Science) and corresponding precast 2% agarose gel cassette (Sage Science) as per the manufacturer's protocol. A second hemi-nested PCR amplification with indexing was performed, followed by another PCR purification and quantification using dsDNA Broad Range Qubit Kit (Life Technologies). Samples were shipped to Macrogen for quality control, further processing, and sequencing using HiSeq 2500 (Illumina).