We describe here a technique that combines transposon mutagenesis with high-throughput sequencing to identify and quantify transposon leptospiral mutants in tissues after a challenge of hamsters. This protocol can be used to screen mutants for survival and dissemination in animals and can also be applied to in vitro studies.
In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness during infection of Golden Syrian hamsters. Tn-Seq combines random transposon mutagenesis with the power of high-throughput sequencing technology. Animals are challenged with a pool of transposon mutants (input pool), followed by harvesting of blood and tissues a few days later to identify and quantify the number of mutants in each organ (output pools). The output pools are compared to the input pool to evaluate the in vivo fitness of each mutant. This approach enables screening of a large pool of mutants in a limited number of animals. With minor modifications, this protocol can be performed with any animal model of leptospirosis, reservoir host models such as rats and acute infection models such as hamsters, as well as in vitro studies. Tn-Seq provides a powerful tool to screen for mutants with in vivo and in vitro fitness defects.
Identification of virulence genes for some bacteria, such as Leptospira spp., is difficult because of the limited number of genetic tools available. One commonly used approach is the creation of a collection of mutants by random transposon mutagenesis followed by the identification of the insertion site in each mutant and virulence testing of individual transposon mutants in an animal model. This approach is time-consuming, expensive, and requires a large number of animals.
When random mutagenesis was first developed for the pathogen Leptospira interrogans, genes involved in virulence were identified by testing individual mutants in an animal model1. Mutants were selected based on criteria such as their potential roles in signaling or motility or their predicted outer membrane or surface location. As the majority of leptospiral genes encode hypothetical proteins of unknown function2, selecting mutants based on these criteria limits the ability to discover novel leptospiral virulence genes.
More recently, pools of L. interrogans transposon mutants were screened for infectivity in the hamster and mouse models3. Each animal was challenged with a pool of up to 10 mutants. Infectivity of a mutant was scored as positive if it was detected by PCR of cultures obtained from blood and kidney. PCR testing was laborious because it required an individual PCR reaction for each mutant in the pool. Because the frequency of each mutant in the cultures was not quantified, the approach was biased towards identification of highly attenuated mutants.
We describe a transposon sequencing (Tn-Seq) technique, as a strategy to more efficiently screen for virulence genes. Tn-seq consists of the creation of a library of mutants by transposon mutagenesis followed by massive parallel sequencing4,5,6. Briefly, transposon mutants are pooled, inoculated into animals, and later recovered from different organs (output pools). The DNA from the output pools is extracted and digested with restriction enzymes or sheared by sonication. Two rounds of PCR targeting the junctions of the transposon insertion sites are performed. This step enables the addition of the adaptors necessary for the sequencing. The resulting PCR products are analyzed by high-throughput sequencing to identify the transposon insertion site of each mutant of the pool along with their relative abundance, which is compared to the initial composition of the pool of mutant.
The primary advantage of this approach is the ability to simultaneously screen a large number of mutants with a small number of animals. Tn-Seq does not require the prior knowledge of the transposon insertion sites which increases the chances of discovering new Leptospira-specific genes involved in virulence with less time and greater efficiency. Because leptospiral burden in tissues is relatively high in rodent models susceptible to lethal infection (typically 104 to 108 bacteria/g of tissue)7,8,9 as well as in reservoir hosts10,11, tissues can be directly analyzed without the need to culture, reducing biases due to in vitro growth.
In Tn-Seq studies with most bacterial pathogens described to date, the high frequency of insertional mutagenesis allowed infection with large pools containing mutants collectively having multiple closely-spaced transposon insertions within every gene4,12,13,14. Tn-Seq has also been developed for a bacterium for which the mutagenesis frequency is much lower6. With Leptospira, a library of transposon mutants can be generated by introducing the transposon on a mobilizable plasmid by conjugation as described by Slamti et al15. However, the frequency of transposon mutagenesis of L. interrogans is low. When the Himar1 transposon was introduced on a conjugative plasmid, the transconjugant frequency was reported to be only 8.5 x 10-8 per recipient cell with the Lai strain of L. interrogans16 and is likely to be similarly poor with most other strains of L. interrogans. The protocol described here is in part based on that developed for Borrelia burgdorferi, in which the frequency of transposon insertional mutagenesis is also low6.
For our pilot experiment with the protocol17, we conducted transposon mutagenesis with L. interrogans serovar Manilae strain L495 because of the success of other groups in isolating transposon insertion mutants in the strain along with its low LD50 (lethal dose) for virulence1. We screened 42 mutants by Tn-Seq and identified several mutant candidates defective in virulence, including two with insertions in a candidate adenylate cyclase gene. Individual testing of the two mutants in hamsters confirmed that they were deficient in virulence17.
CAUTION: Pathogenic strains of Leptospira spp. must be handled under Biosafety Level 2 (BSL-2) containment procedures. Appropriate personal protective equipment (PPE) must be worn. A Class II biosafety cabinet must be used for all manipulations of pathogenic Leptospira spp.
1. Creation of the Transposon Mutant Library15
2. Animal Experiment (Figure 3)
3. Construction of Genomic Libraries for High-throughput Sequencing (Figure 4)
4. High-throughput Sequencing and Data Analysis
Creation of a library of transposon mutants in L. interrogans by conjugation requires a filtration unit, as shown in Figure 1. We recovered 100-200 transconjugants from each mating.
The transposon insertion site is identified in each mutant by sequencing the PCR product generated by semi-random PCR that targets the end of the transposon and adjacent host sequences15 (Figure 2A). An example of results of the semi-random PCR is shown in Figure 2B. In most cases a dominant amplicon will be observed when the Deg1 and Tnk1 primers are used for the first round of PCR and the Tag and TnkN1 for the second round. However, due to the low specificity of the pentameric sequence (…N10GATAT-3') at the 3' end of the Deg1 primer, this primer will anneal at multiple locations throughout the leptospiral genome. If these sites are present near the transposon end, multiple PCR products may be obtained. When multiple amplicons are generated, they can be purified together from the PCR mix and sequenced directly; gel purification of each amplicon is not necessary because they will all contain the same sequence downstream of where TnkN1 primer anneals. On the other hand, PCR may fail if the nearest copy of the sequence targeted by the Deg1 primer is located at a great distance from the transposon end. If no PCR products are detected, the first round of semi-random PCR can be repeated with the Deg1 primer and Tnk2 primer, which targets the opposite end of the transposon. In this case TnkN2 and Tag would be used for the second round; the amplicon would be sequenced with the TnkN2 primer (Figure 2B). Alternatively, the first round of PCR can be done with the Deg2 primer, whose 3' end (…N10TCTT-3') targets a four- rather than a five-nucleotide sequence. Four different sets of primers can be used for the first round of PCR (PCR #1): Tnk1+Deg1, Tnk1+Deg2, Tnk2+Deg1, Tnk2+Deg2. When one set of primers does not work, use another set. If this second set also fails, use another one and so on. Spontaneous kanamycin resistant mutants are very rare, and arise at a frequency of <10-10 34.
During the preparation of genomic libraries (Figure 4), a couple of steps can be verified. Shearing the DNA by sonication should be confirmed by electrophoresis of an aliquot in an agarose gel (Figure 5). When DNA is sheared correctly, DNA fragments ranging from 200 to 600 bp will form a smear in a 2% agarose gel. Before sending libraries for high-throughput sequencing, the generation of PCR products by nested PCR reactions must to be confirmed by agarose gel electrophoresis (Figure 6).
After processing the sequencing reads following the protocol described here (section 4.2.), the frequency of each mutant in the input pool and in all tissues is determined using the equation in section 4.3.3. The output/input ratio for each mutant in each tissue is calculated with the equation in section 4.3.4. Figure 7 shows an example of results obtained with the Tn-Seq approach. Alternatively, the MAGenTA tool can be used to process and analyze the sequencing data 31. Mutants with statistically significantly reduced and increased fitness were identified. Mutations in known leptospiral virulence genes caused reduced fitness, validating the Tn-Seq approach.
Figure 1: Filtration unit used for conjugation. The filtration unit is assembled by inserting the stopper of the filter support into a side-arm Erlenmeyer flask, positioning the acetate-cellulose filter onto the base with sterile forceps, then placing the funnel onto the base, and securing the system with a clamp. The filtration unit is then connected to the vacuum system. Please click here to view a larger version of this figure.
Figure 2: Identification of the transposon insertion sites. (A) Scheme showing annealing sites of semi-random PCR primers. Tnk1 and Tnk2 primers anneal close to the opposite ends of the transposon (Tn). Deg1 is a 35-nucleotide primer that contains a stretch of 10 degenerate nucleotides followed by the sequence GATAT at the 3' end. The first round of PCR (PCR #1) is conducted with Deg1 and Tnk1 or with Deg1 and Tnk2. TnkN1 and TnkN2 primers, used for PCR #2, anneal between the transposon ends and Tnk1 and Tnk2, respectively. The sequence of the Tag primer is identical to that of the 5' end of the Deg1 primer. (B) Example of agarose gel obtained after the second round of PCR with 14 transposon mutants (lanes 1 to 14). The first round of PCR was performed with Deg1 and Tnk1; the second round was done with Tag and TnkN1. Positive PCR is represented by "+" and a negative PCR by "-". "KmR" represents the kanamycin resistance cassette. Please click here to view a larger version of this figure.
Figure 3: Flow chart for obtaining the output pool. On the day of challenge (day 0), each leptospiral mutant is counted by darkfield microscopy, diluted to the same density, and combined to form the input pool (details provided in section 2.1). Hamsters are challenged intraperitoneally with the input pool. In addition, three cultures were started with the input pool. On the day of inoculation (day 0) and when the cultures reach a density of ≈ 108/mL, the input pool and cultures are collected by centrifugation and stored as pellets at -80 °C until use (see section 2.3). On the endpoint day (day preselected to terminate the animals, e.g., day 4 after challenge), animals are euthanized; blood, kidney, and liver are collected and stored at -80 °C until use (see section 2.4). Please click here to view a larger version of this figure.
Figure 4: Flow chart of the genomic library preparation. (A) DNA is extracted from blood, tissues or cultures following the protocol described in section 3.1. The red box represents the transposon (Tn). (B) The DNA is sheared by sonication into fragments between 200 and 600 bp. Further details are provided in section 3.2. (C) C-tails (yellow boxes) are added to all DNA fragments with terminal deoxynucleotidyl transferase (TdT) as described in section 3.3 and Table 4. The genomic library is prepared for sequencing by nested PCR (D-E). The first round of PCR is performed with TnkN3 and olj376 primers specific to the transposon and the C-tail, respectively. See Table 3 and 5. Only fragments containing the transposon ends (red box) are amplified. Note: Use 3 times more olj376 primer than TnkN3 because all DNA fragments have C-tails. The second round of PCR is conducted with pMargent2, specific for the transposon end, and one indexing primer, containing a six-base-pair barcode sequence (in pink) allowing all samples to be multiplexed in a single sequencing lane. See Table 3 and 6. Both primers contain sequences necessary for binding the flow-cell during Illumina sequencing (green and purple box). (F) The resulting PCR products are cleaned, and then their concentration is measured as described in section 3.6. (G) All genomic libraries are pooled together; each library has a different barcode and (H) the pool is sent to the sequencing platform. Please click here to view a larger version of this figure.
Figure 5: Sonication of DNA. DNA was purified from the livers of eight infected hamster (lanes 1 to 8), sonicated, and examined by electrophoresis in a 2% agarose gel. Sheared DNA is characterized by a smear in which the size of most of the fragments is between 200 and 600 bp. Please click here to view a larger version of this figure.
Figure 6: Genomic libraries for high-throughput sequencing. Genomic libraries were prepared by ested PCR with DNA from the blood of eight infected hamsters (lanes 1 to 8) and examined by electrophoresis in a 2% agarose gel. The size of most of the PCR products is between 200 and 600bp. Negative control (no TnkN3 primer in PCR #1 mix, see Table 5) shows no amplification (not shown). Please click here to view a larger version of this figure.
Figure 7: Fitness of mutants from Tn-Seq experiment. Fitness of a pool of mutants in hamster's kidney 4 days after challenge (adapted from Lourdault's study17). The output/input ratio of all 42 mutants was determined for each animal. Each mutant is named by the gene (lic) or the intergenic region (inter) the transposon is inserted into or the name commonly used in the literature. Each ratio is represented by a black diamond. The median of ratios (red line) was determined for each mutant and compared to 1.0 using the Wilcoxon rank test. The dotted line represents fitness of 1.0, which corresponds to neutral fitness. Mutants whose fitness is significantly affected are marked by asterisks: * P <0.05; ** P <0.01. Please click here to view a larger version of this figure.
Reagents | Volume for one reaction |
Master mix 2X | 12.5 µL |
Primer 1, 10 mM (TnK1 or TnK2)* | 1.2 µL |
Primer 2, 10 mM (Deg1 or Deg2)* | 1.2 µL |
Water | 8.8 µL |
Template (cells lysate or DNA) | 1.3 µL |
Final volume | 25 µL |
* Primer sequences in Table 3. |
Table 1: Identification of the transposon insertion site, semi-random PCR #1 mix.
Reagents | Volume for one reaction |
Master mix 2X | 12.5 µL |
Primer 1, 100 mM (TnKN1 or TnKN2)* | 0.2 µL |
Primer 2, 100 mM (Tag)* | 0.2 µL |
Water | 10.1 µL |
PCR products from PCR#1 | 0.8 µL |
Final volume | 25 µL |
* Primer sequences in Table 3. |
Table 2: Identification of the transposon insertion site, semi-random PCR #2 mix.
Name | Sequence (5'-3') | Reference | ||||
Primers used for semi-random PCR | ||||||
Tnk1 | CTTGTCATCGTCATCCTTG | 15 | ||||
Tnk2 | GTGGCTTTATTGATCTTGGG | |||||
Deg1 | GGCCACGCGTCGACTAGTACNNNNNNNNNNGATAT | |||||
Deg2 | GGCCACGCGTCGACTAGTACNNNNNNNNNNTCTT | |||||
TnkN1 | CGTCATGGTCTTTGTAGTCTATGG | |||||
TnKN2 | TGGGGATCAAGCCTGATTGGG | |||||
Tag | GGCCACGCGTCGACTAGTAC | |||||
Primers used for Illumina sequencing | ||||||
TnkN3 | CGGGGAAGAACAGTATGTCGAGCTATTTTTTGACTTACTGGGGATCAAGCCTGATTGGG | 17 | ||||
olj376 | GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGGGGGGGGGGGGGGG | 6 | ||||
pMargent2 | AATGATACGGCGACCACCGAGATCTACACTCTTTCCGGGGACTTATCAGCCAACCTGTTA | |||||
IP 1 | CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | Illumina sequencing (barcodes in bold) | ||||
IP 2 | CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |||||
IP 3 | CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |||||
IP 4 | CAAGCAGAAGACGGCATACGAGATTGGTCAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |||||
IP 5 | CAAGCAGAAGACGGCATACGAGATCACTGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |||||
IP 6 | CAAGCAGAAGACGGCATACGAGATATTGGCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |||||
IP 7 | CAAGCAGAAGACGGCATACGAGATGATCTGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |||||
IP 8 | CAAGCAGAAGACGGCATACGAGATTCAAGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |||||
IP 9 | CAAGCAGAAGACGGCATACGAGATCTGATCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |||||
IP 10 | CAAGCAGAAGACGGCATACGAGATAAGCTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |||||
IP 11 | CAAGCAGAAGACGGCATACGAGATGTAGCCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |||||
IP 12 | CAAGCAGAAGACGGCATACGAGATTACAAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |||||
IP 13 | CAAGCAGAAGACGGCATACGAGATTTGACTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |||||
IP 14 | CAAGCAGAAGACGGCATACGAGATGGAACTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |||||
IP 15 | CAAGCAGAAGACGGCATACGAGATTGACATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |||||
IP 16 | CAAGCAGAAGACGGCATACGAGATGGACGGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |||||
IP 17 | CAAGCAGAAGACGGCATACGAGATCTCTACGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |||||
IP 18 | CAAGCAGAAGACGGCATACGAGATGCGGACGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |||||
IP 19 | CAAGCAGAAGACGGCATACGAGATTTTCACGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |||||
IP 20 | CAAGCAGAAGACGGCATACGAGATGGCCACGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |||||
IP 21 | CAAGCAGAAGACGGCATACGAGATCGAAACGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |||||
IP 22 | CAAGCAGAAGACGGCATACGAGATCGTACGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |||||
IP 23 | CAAGCAGAAGACGGCATACGAGATCCACTCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |||||
IP 24 | CAAGCAGAAGACGGCATACGAGATGCTACCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |||||
IP 25 | CAAGCAGAAGACGGCATACGAGATATCAGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |||||
IP 26 | CAAGCAGAAGACGGCATACGAGATGCTCATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |||||
IP 27 | CAAGCAGAAGACGGCATACGAGATAGGAATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |||||
IP 28 | CAAGCAGAAGACGGCATACGAGATCTTTTGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |||||
IP 29 | CAAGCAGAAGACGGCATACGAGATTAGTTGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT | |||||
IP seq | GATCGGAAGAGCACACGTCTGAACTCCAGTCAC | |||||
pMargent3 | ACACTCTTTCCGGGGACTTATCAGCCAACCTGTTA | 6 |
Table 3: Primer sequences.
Reagents | Volume for one reaction |
Sheared DNA | 500 ng (see equation in 3.3.2.) |
Water | Up to 14.5 µL |
5x TdT buffer | 4 µL |
(9.5 mM dCTP + 0.5 mM ddCTP) mix* | 1 µL |
TdT (30 U/µL) | 0.5 µL |
Final volume | 20 µL |
* Mix 3 µLof 10 mM ddCTP, 5.7 µL of 100 mM dCTP and 51.3 µL of water |
Table 4: C-tailing reaction with terminal deoxynucleotidyl transferase (TdT).
Reagents | Volume for one reaction | |
Library reaction | Control reaction | |
Master mix 2x | 12.5 µL | 12.5 µL |
Primer 1, 30 µM (TnkN3)* | 0.5 µL | – |
Primer 2, 30 µM (olj376)* | 1.5 µL | 1.5 |
Water | 7.5 µL | 8 µL |
C-tailed DNA | 3 µL | 3 µL |
Final volume | 25 µL | 25 µL |
* Primer sequences in Table 3. |
Table 5: Construction of genomic library, PCR #1 mix.
Reagents | Volume for one reaction | |
Library reaction | Control reaction | |
Master mix 2x | 25 µL | 12.5 µL |
Primer 1, 30 µM (pMargent2)* | 0.5 µL | 0.25 µL |
Primer 2, 30 µM (indexing primer)* | 0.5 µL | 0.25 µL |
Water | 23 µL | 11.5 µL |
PCR products from PCR#1 | 1 µL | 0.5 µL |
Final volume | 50 µL | 25 µL |
* Primer sequences in Table 3. |
Table 6: Construction of genomic library, PCR #2 mix.
Although results from our pilot experiment for hamster challenged intraperitoneally with 42 L. interrogans mutants are presented17, we expect that larger pools of mutants can be screened by Tn-Seq. Because the frequency of transconjugants is low (100-200 transconjugants/mating), several matings are necessary to generate a sufficient number of mutants for large Tn-Seq experiments. Maintaining a large number of mutants in liquid cultures presents logistical challenges that must be addressed. Cultures can be incubated in deep 96-well plates. Culture densities can be monitored by optical density readings in a spectrophotometer set to 420 nm. The determination of the number of animals to use depends in part on the size of the input pool and must be determined by power analysis. For large-scale experiments, we recommend that the power analysis be performed in consultation with a biostatistician.
Bottlenecks can impact the recovery of random mutants from the output pool. Although severe bottlenecks were not observed in our pilot study17 (Figure 7), large-scale experiments may be affected by random loss of mutants. Increasing the challenge dose by increasing the number of cells per mutant in the input pool will minimize the likelihood of bottlenecks35. The newly published Galaxy tool MAGenTA provides the necessary tool to assess bottlenecks and fitness31.
Tn-Seq experiments planned for an alternative route of infection, other Leptospira strains, or other rodent models require additional considerations. If the kinetics of infection in the host species is not known or is not continuously exponential during the course of infection within each tissue to be examined, DNA should be obtained from culturing of the tissues rather than directly from the tissues. This additional step will minimize overestimates of mutant fitness due to detection of DNA from dead spirochetes. If the output pool is obtained from culturing the tissues, the input pool should be cultured to address effects of in vitro growth. We also recommend a pilot experiment with a small number of mutants to determine whether bottlenecks will be a concern and to aid the power analysis for large-scale experiments.
Confirmation of altered fitness as determined by Tn-Seq will require testing of individual mutants in the animal model. Currently, each mutant must be sequenced individually prior to Tn-Seq to identify the mutant carrying the insertion in the gene of interest. However, if allele replacement in pathogenic Leptospira becomes easy, sequencing of individual mutants will no longer be necessary. Alternatively, transcription activator-like effectors have been successfully used to diminish lig genes expression in L. interrogans36. This technique could be used to down-regulate genes disrupted in mutants with altered fitness identified by Tn-Seq.
When interpreting the Tn-Seq data, interactions between bacteria should to be taken into account. It is theoretically possible that a decrease in fitness could be due to competition between mutants rather than a direct effect of the transposon insertion. Additionally, an absence of change in in vivo fitness of a mutant in a pool could be due to cooperation between mutants. For example, a mutant that fails to produce an essential factor could be complemented intercellularly by production of the factor by other mutants in the pool. We observed an increase in fitness for several mutants, which may be a consequence of the reduced metabolic burden of no longer synthesizing proteins that are not essential for growth, as shown for Salmonella enterica37.
This protocol can be used to identify genes involved in metabolism or survival under stressful conditions in vitro 38,39. For example, growing Leptospira in different conditions like high sodium chloride concentration, limited iron, and acidic pH could identify genes responsible for acidic survival or stress resistance. These in vitro experiments can also be performed with saprophytic strains such as L. biflexa strain Patoc I because this Tn-Seq method can be applied to all sequenced Leptospira strains.
The authors have nothing to disclose.
This work was supported by a Veterans Affairs Merit Award (to D.A.H.) and a National Institute of Health grant R01 AI 034431 (to D.A.H.).
Kanamycin sulfate from Streptomyces kanamyceticus | Sigma-Aldrich | K4000 | |
2,6-diaminopimelic acid | Sigma-Aldrich | D1377 | |
Spectinomycin dihydrochloride pentahydrate | Sigma-Aldrich | S4014 | |
Axio Lab A1 microscope with a darkfieldcondenser | Zeiss | 490950-001-000 | |
DNeasy blood and tissue kit | Qiagen | 69504/69506 | |
MinElute PCR Purification | Qiagen | 28004/28006 | |
QIAquick PCR purification kit | Qiagen | 28104/28106 | |
Model 505 Sonic Dismembrator | Fisher Scientific | FB-505 | |
2.5" Cup horn | Fisher Scientific | FB-4625 | |
Bead Ruptor 24 | Omni International | 19-010 | Step 3.1.2.4 |
Terminal deoxynucleotidyl transferase | Promega | M828C | |
Master mix Phusion | Thermo Scientific | F531 | Preparation of genomic libraries, step 3.4. |
DreamTaq Master Mix | Thermo Scientific | K9011/K9012 | Identification of the transposon insertion site, step 1.2. |
dCTP | Thermo Scientific | R0151 | |
ddCTP | Affymetrix/ USBProducts | 77112 | |
T100 Thermal cycler | BioRad | 1861096 | |
Qubit 2.0 fluorometer | Invitrogen | Q32866 | step 3.6. |
Qubit dsDNA HS assay kit | Invitrogen | Q32851/Q32854 | step 3.6. |
Qubit assay tubes | life technologies | Q32856 | step 3.6. |
PBS pH 7.2 (1X) | Gibco | 20012-027 20012-050 | |
Disposable scalpel No10 | Feather | 2975#10 | |
Plastic K2 EDTA 2 ml tubes | BD vacutainer | 367841 | |
syringe U-100 with 26G x ½” needle | BD vacutainer | 329652 | IP challenge, step 2.2.1. |
3 mL Luer-Lok tip syringe | BD vacutainer | 309657 | Cardiac puncture, step 2.4.2. |
25G X 5/8” needle | BD vacutainer | 305901 | Cardiac puncture, step 2.4.2. |
25 mm fritted glass base with stopper | EMD Millipore | XX1002502 | Filtration unit system, step 1.1.7. |
25 mm aluminum spring clamp | EMD Millipore | XX1002503 | Filtration unit system, step 1.1.7. |
15 ml borosilcate glass funnel | EMD Millipore | XX1002514 | Filtration unit system, step 1.1.7. |
125 ml side-arm Erlenmeyer flask | EMD Millipore | XX1002505 | Filtration unit system, step 1.1.7. |
Acetate-cellulose filter VVPP (pore size 0.1 mm; diameter 25 mm) | EMD Millipore | VVLP02500 |