TnSeq raw data

Transposon library construction, genomic DNA Extraction, and sequencing.

Transposon library after buoyancy centrifugation was collected and resuspended in 400ul 10mM Tris (pH=9). After beads-beating, genomic DNA was extracted by Phenol-Chloroform method. DNA concentration was measured and quantified by Nanodrop and Qubit. For building transposon sequencing library, approximately 5ug genomic DNA was resuspended in 150ul TE buffer and transferred to a Covaris tube, Genomic DNA was disrupted to 200-500bp size range by sonication with the following parameters: duty cycle (10%), intensity (4), cycles/burst (200), time (80s). The fragmented genomic DNA was size-selected and purified by AMPure XP beads. The fragmented genomic DNA was further subjected to end repair and dA tailing. Annealed adapter was ligated to the dA tailed fragmented genomic DNA and the linked ligated DNA fragment was used as template of 1^st round nested PCR to amplify fragments containing adaptor and transposon junction. Indexed barcoded sequencing and illumina sequencing adaptor was added by 2^nd nested PCR. All sequence libraries were examined by Agilent 2100 Bioanalyzer and subjected to next generation sequencing.

Transposon mutagenesis.

Mycobacterium smegmatis mc²155 strain was grown to stationary phase (OD>6) in 50ml of 7H9 growth medium. Bacterial cultures were washed and resuspended in 5ml MP buffer (50mM Tris, 150mM NaCl, 10mM MgSO4, 2mM CaCl2). To transduce bacteria with MycoMarT7 phage, approximately 10¹¹ plaque forming units of phage (PFU) was added to the bacterial suspension in MP buffer and incubated at 37°C for 4h. Immediately after transduction, ~300-400ul of the transduction mixture was plated on 15-cm LB agar plates, containing 20ug/ml kanamycin and 0.1% Tween80. After 3 days, library size was determined, and bacteria was scrapped and stored in 7h9 medium plus 15% glycerol as library stock. The transposon library was made in triplicate. The transposon library was cultured to an OD_600nm of 0.8 and 1ml of sample was loaded onto 10ml of stock isotonic percoll medium, with buoyant density beads as fiducial markers. Buoyancy centrifugation was conducted at 18°C, and spinning at 20k rpm (~50,000 g), for 1h20m. Three buoyancy fractions were isolated: “high” (>1.02 g cm^-3, <1.064 g cm^-3), “middle” (>1.064 g cm^-3, <1.102 g cm^-3), and “low” (>1.102 g cm^-3). Three biological replicates of the buoyancy centrifugation were conducted for the transposon library made in triplicate each of the three transposon libraries: 3 (libraries) x 3 (buoyancy centrifugation experiments) x 3 (buoyancy fractions) = 27 individual samples.

Transposon mapping and analysis.

Reads processing and TA loci mapping were performed through software TRANSIT ²⁶. Loci that were differentially disrupted by transposon were analysed using resampling test in TRANSIT with default parameter. Different buoyancy fractions were compared with input libraries and genes that are over-represented (log2FC < -1, adjusted p-value < 0.05) and under-represented (Log2FC >1, p-value < 0.05) were plotted.