recovered from different tissues. DNA extracted from cultures or
tissues is then sheared by sonication. DNA fragments are processed
by two PCR steps that amplify sequences across one end of the
transposon insertion site. The resulting genomic library is then
analyzed by Illumina sequencing.
Here, we describe the steps to prepare genomic libraries for
high-throughput sequencing. The sequencing reactions generate
FASTQ files, which can be analyzed to determine the counts for
each mutant in the pool [7].
2 Materials
2.1 Preparation
of the Pool of Mutants
and In Vitro or In Vivo
Testing
1. Leptospira Himar1 mariner transposon mutants (see Note 1).
2. EMJH basal medium [8, 9]: Dissolve 1 g Na 2 HPO 4 , 0.3 g
KH 2 PO 4 , and 1 g NaCl in 998 ml sterile distilled water. Add
the following stock solutions prepared in sterile distilled water:
1 ml 10% (v/v) glycerol and 1 ml 25% (w/v) NH 4 Cl. Measure
the pH, which should be 7.4. Sterilize by autoclaving.
3. EMJH supplement: Dissolve 10 g bovine serum albumin
(BSA) in 50 ml sterile distilled water with slow and constant
stirring. Once the BSA is dissolved, add the following stock
solutions prepared in sterile distilled water (stock solutions
should be filtered a 0.22 μM filter): 1 ml thiamine (stock
5 mg/ml thiamine chloride), 1 ml calcium chloride (stock
10 mg/ml CaCl 2
. 2H 2 O), 1 ml magnesium chloride (stock
10 mg/ml MgCl 2
. 6H 2 O), 1 ml zinc sulfate (stock 4 mg/ml
ZnSO 4
. 7H 2 O), 0.1 ml manganese sulfate (stock 3 mg/ml
MnSO 4
. H 2 O), 10 ml ferrous sulfate (stock 5 mg/ml FeSO 4
. 7H 2 O), 1 ml vitamin B 12 (stock 0.2 mg/ml vitamin B 12 ),
12.5 ml Tween 80 (make fresh, 10 ml Tween 80 in 90 ml
distilled water). Adjust pH to 7.4 with NaOH, and then bring
volume to 100 ml with sterile distilled water. Sterilize by filtration through a 0.22 μM filter.
4. EMJH liquid medium: Add 100 ml EMJH supplement to
900 ml EMJH basal medium. Add 1 g lactalbumin hydrolysate,
100 μl superoxide dismutase (stock 10 mg/ml superoxide dismutase in PBS, store aliquots at À20
C), and 0.04 g sodium
pyruvate. Filter sterilize (0.22 μM filter). Aliquot into smaller
volumes and store at 4
C.
5. Kanamycin: 50 mg/ml solution in water.
6. 50 ml centrifuge tubes.
7. 125 ml screw-capped Erlenmeyer flasks.
8. Shaking incubator.
9. Dark-field microscope.
98
Kristel Lourdault and James Matsunaga
tissues is then sheared by sonication. DNA fragments are processed
by two PCR steps that amplify sequences across one end of the
transposon insertion site. The resulting genomic library is then
analyzed by Illumina sequencing.
Here, we describe the steps to prepare genomic libraries for
high-throughput sequencing. The sequencing reactions generate
FASTQ files, which can be analyzed to determine the counts for
each mutant in the pool [7].
2 Materials
2.1 Preparation
of the Pool of Mutants
and In Vitro or In Vivo
Testing
1. Leptospira Himar1 mariner transposon mutants (see Note 1).
2. EMJH basal medium [8, 9]: Dissolve 1 g Na 2 HPO 4 , 0.3 g
KH 2 PO 4 , and 1 g NaCl in 998 ml sterile distilled water. Add
the following stock solutions prepared in sterile distilled water:
1 ml 10% (v/v) glycerol and 1 ml 25% (w/v) NH 4 Cl. Measure
the pH, which should be 7.4. Sterilize by autoclaving.
3. EMJH supplement: Dissolve 10 g bovine serum albumin
(BSA) in 50 ml sterile distilled water with slow and constant
stirring. Once the BSA is dissolved, add the following stock
solutions prepared in sterile distilled water (stock solutions
should be filtered a 0.22 μM filter): 1 ml thiamine (stock
5 mg/ml thiamine chloride), 1 ml calcium chloride (stock
10 mg/ml CaCl 2
. 2H 2 O), 1 ml magnesium chloride (stock
10 mg/ml MgCl 2
. 6H 2 O), 1 ml zinc sulfate (stock 4 mg/ml
ZnSO 4
. 7H 2 O), 0.1 ml manganese sulfate (stock 3 mg/ml
MnSO 4
. H 2 O), 10 ml ferrous sulfate (stock 5 mg/ml FeSO 4
. 7H 2 O), 1 ml vitamin B 12 (stock 0.2 mg/ml vitamin B 12 ),
12.5 ml Tween 80 (make fresh, 10 ml Tween 80 in 90 ml
distilled water). Adjust pH to 7.4 with NaOH, and then bring
volume to 100 ml with sterile distilled water. Sterilize by filtration through a 0.22 μM filter.
4. EMJH liquid medium: Add 100 ml EMJH supplement to
900 ml EMJH basal medium. Add 1 g lactalbumin hydrolysate,
100 μl superoxide dismutase (stock 10 mg/ml superoxide dismutase in PBS, store aliquots at À20
C), and 0.04 g sodium
pyruvate. Filter sterilize (0.22 μM filter). Aliquot into smaller
volumes and store at 4
C.
5. Kanamycin: 50 mg/ml solution in water.
6. 50 ml centrifuge tubes.
7. 125 ml screw-capped Erlenmeyer flasks.
8. Shaking incubator.
9. Dark-field microscope.
98
Kristel Lourdault and James Matsunaga