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 1st round nested PCR to amplify fragments
containing adaptor and transposon junction. Indexed barcoded sequencing and
illumina sequencing adaptor was added by 2nd 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 OD600nm
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.

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