Tn-seq library 3 hours after vancomycin treatment
Frozen cell pellets were resuspended in 500 L lysis buffer with lysozyme and RNase A (20 mM Tris-HCl pH 7.5, 50 mM EDTA, 100 mM NaCl, 2 mg/mL lysozyme, 120 g/mL RNase A) and incubated at 37C for 20-30 min. Next, the incubated cell lysate was mixed with 60 L 10% N-lauroylsarkosine and further incubated at 37C for 15 min. Genomic DNA (gDNA) was purified using 600 L phenol, then 600 L phenol:chloroform:isoamyl alcohol (25:24:1), and finally 600 L pure chloroform. DNA in the aqueous phase was precipitated using 1/10 volumes of 3M NaOAc and 2 volumes of 100% ethanol. DNA pellet was washed with 70% ethanol, air-dried on bench, and resuspended in 10 mM Tris-HCl, pH 8.5 and stored at 4C. For each sample, 6 g of DNA was used for MmeI digestion in 200 L (6 g gDNA, 6 L MmeI (2000 U/mL, NEB), 0.5 L 32 mM S-adenosylmethionine, 20 L NEB CutSmart Buffer, and ddH2O up to 200 L). DNA was digested for 2.5 h at 37C, after which 2 L calf intestinal phosphatase (10,000 U/mL, NEB) was added to the digest and incubated for 1h at 37C. Digested gDNA was extracted with 200 L phenol:chloroform:isoamyl alcohol (25:24:1) followed by 200 L of pure chloroform. DNA in the aqueous phase was first mixed with 1/10 volume of 3M NaOAc and 67 ng/mL glycogen, and then 2.5 volumes of 100% ethanol. The tubes were then placed at -80C for 20 min and then centrifuged at max speed for 15 min at 4C. Precipitated DNA was washed with 150 L 70% ethanol twice at room temperature, air-dried, and resuspended in 15 L ddH2O.      For annealing of the DNA adaptor, 20 L of 100 M synthesized oligos (IDT) were mixed with 1 L of 41 mM Tris-HCl pH 8.0 (final concentration of adaptor: 50 M in 1mM Tris-HCl pH 8.0). Oligos were annealed by heat denaturation (95 C for 5 min) and stepwise cool-down (94C for 45 s then repeat with -0.3C per cycle for 250 cycles, then hold at 15C) using PCR machine. Annealed adaptors were diluted to 3.3 M in ddH2O and stored at -20C.For adaptor ligation, 5 L of digested DNA were mixed with 1 L of 3.3 M DNA adaptor, 1 L of 10x T4 DNA ligase buffer (NEB), 1 L of T4 DNA ligase (400,000U/mL, NEB) and 2 L ddH2O. The ligation mix was incubated overnight at 16C in a PCR machine.Amplification of adaptor-ligated DNA library was performed using barcoded primers and Phusion high-fidelity DNA polymerase (NEB) for 18 cycles according to provided instructions. The PCR products were mixed in equal amounts, purified by size-exclusion, and submitted for sequencing using Illumina sequencing primer (5-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3). Deep sequencing was done using Illumina HiSeq 2500 (Illumina) by the University of Michigan DNA Sequencing Core.