TnSeq raw data

<b>Transposon library construction, genomic DNA Extraction, and sequencing.</b> 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. <b>Transposon mutagenesis.</b><b></b> <i>Mycobacterium smegmatis</i> mc²155 strain was grown to stationary phase (OD&gt;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” (&gt;1.02 g cm^-3, &lt;1.064 g cm^-3), “middle” (&gt;1.064 g cm^-3, &lt;1.102 g cm^-3), and “low” (&gt;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. <b>Transposon mapping and analysis.</b><b></b> 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 &lt; -1, adjusted p-value &lt; 0.05) and under-represented (Log2FC &gt;1, p-value &lt; 0.05) were plotted. @font-face {font-family:"Cambria Math"; panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic-font-family:roman; mso-font-pitch:variable; mso-font-signature:-536870145 1107305727 0 0 415 0;}@font-face {font-family:Calibri; panose-1:2 15 5 2 2 2 4 3 2 4; mso-font-charset:0; mso-generic-font-family:swiss; mso-font-pitch:variable; mso-font-signature:-536859905 -1073732485 9 0 511 0;}p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-unhide:no; mso-style-qformat:yes; mso-style-parent:""; margin:0in; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Calibri",sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:Calibri; mso-fareast-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Arial; mso-bidi-theme-font:minor-bidi; mso-ansi-language:FR; mso-fareast-language:EN-US;}.MsoChpDefault {mso-style-type:export-only; mso-default-props:yes; font-family:"Calibri",sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:Calibri; mso-fareast-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Arial; mso-bidi-theme-font:minor-bidi; mso-ansi-language:FR; mso-fareast-language:EN-US;}div.WordSection1 {page:WordSection1;}

**转座子文库构建、基因组DNA提取与测序**
经浮力离心（buoyancy centrifugation）回收转座子文库，并重悬于400 μL 10 mM Tris-HCl缓冲液（pH 9.0）。经珠磨法破碎后，采用酚-氯仿抽提法提取基因组DNA。通过Nanodrop分光光度计与Qubit荧光定量仪对DNA浓度进行测定与定量。
构建转座子测序文库时，取约5 μg基因组DNA重悬于150 μL TE缓冲液（TE buffer）中，转移至Covaris超声管（Covaris tube），采用超声破碎法将基因组DNA打断至200~500 bp，参数设置如下：占空比10%、强度4、单次循环脉冲数200、总时长80秒。将打断后的基因组DNA通过AMPure XP磁珠（AMPure XP beads）进行片段大小筛选与纯化。随后对纯化后的片段进行末端修复与dA尾加尾（end repair and dA tailing）。将退火接头（Annealed adapter）与加有dA尾的片段进行连接，以连接产物为模板进行第一轮巢式PCR（nested PCR），扩增包含接头与转座子交界区域的片段。通过第二轮巢式PCR添加带索引条码的Illumina测序接头（indexed barcoded sequencing adaptor）。所有测序文库均通过安捷伦2100生物分析仪（Agilent 2100 Bioanalyzer）进行质检，并用于下一代测序（next generation sequencing）。

**转座子诱变**
将耻垢分枝杆菌（*Mycobacterium smegmatis*）mc²155菌株接种至50 mL 7H9液体培养基中，培养至稳定生长期（光密度值OD>6）。收集细菌培养物，洗涤后重悬于5 mL MP缓冲液（含50 mM Tris、150 mM氯化钠、10 mM硫酸镁、2 mM氯化钙）中。使用MycoMarT7噬菌体（MycoMarT7 phage）进行转导：向MP缓冲液中的细菌悬液加入约10¹¹噬菌斑形成单位（plaque forming units, PFU）的噬菌体，于37 ℃孵育4小时。转导结束后立即取约300~400 μL转导混合液涂布于含20 μg/mL卡那霉素（kanamycin）与0.1%吐温80（Tween80）的15 cm LB琼脂平板（LB agar plates）上。培养3天后，统计文库库容，刮取细菌并重悬于含15%甘油的7H9培养基中，作为文库储备液。本实验共构建3份平行转座子文库。
将转座子文库培养至600 nm光密度值（OD₆₀₀ₙₘ）为0.8，取1 mL样本加入10 mL Percoll分层液（percoll medium）中，以密度梯度标记微球（buoyant density beads）作为参考标记物（fiducial markers）。于18 ℃、20000转/分钟（约50000 g）条件下离心1小时20分钟进行浮力离心。分离得到三个密度组分：“高密”（密度>1.02 g·cm⁻³且<1.064 g·cm⁻³）、“中密”（密度>1.064 g·cm⁻³且<1.102 g·cm⁻³）与“低密”（密度>1.102 g·cm⁻³）。针对3份平行构建的转座子文库，每份文库均进行3次独立的浮力离心实验，最终得到3（文库数）×3（离心重复数）×3（密度组分）=27个独立样本。

**转座子定位与分析**
测序读段处理与TA位点定位通过TRANSIT软件（TRANSIT）完成。采用TRANSIT软件内置的重采样检验（resampling test）默认参数，分析转座子插入突变存在差异的基因位点。将不同密度组分与输入文库进行比较，对富集不足（log₂折叠变化<-1，校正后P值<0.05）与富集过度（log₂折叠变化>1，P值<0.05）的基因进行可视化绘图。