TraDIS method.

TraDIS library preparation protocol
 

Before you start the experiment:
This protocol assumes that the transformation/electroporation conditions were optimised for your strain of interest
 Sterilise 1x 2L conical glass flask
Wash all glassware extremely well to ensure there is no residual detergent, even the smallest amount of residual detergent can dramatically reduce transformation efficiency
 Prepare media for competent cells: For E. coli prepare 500 ml (can go up to one litre if more competent cells are needed) of 2xTY broth, and 20 ml SOC. 
 For washes: Prepare 2L 10% Glycerol, filter sterilise and keep ice cold, but make sure there are no ice crystals when using.
It is easier to split this between 4x 500 ml bottles as you can alternate between a ‘working’ bottle and keeping the others cool in the freezer/fridge/on ice
 Prepare 2L of selection medium with 35 µg/ml Kanamycin 

Day 1:
1. Set up overnight culture:  Inoculate 5 ml LB from single colony from a fresh plate and grow over night at 37 °C
If using a particularly awkward strain, grow O/N culture in 10 ml 2x TY, or increase the volume of overnight culture
2. Pre chill the rotor (JLA-10,500) by storing in the cold room overnight.
3. Prepare 6x sterile 500 ml pots (for the JLA-10,500 rotor) and store them in the cold room overnight.

Day 2: 
4. Add 5 ml of overnight into 2L conical flask containing 500 mL sterile 2x TY broth
Or equivalent 1:100 dilution of your overnight into your day culture of broth
5. Grow cells to an OD600 0.3-0.4 at 37°C, 160 rpm 
This will vary with your strain, use the optimum OD for high transformation efficiency for your strain
6. While cells are growing, make sure all the 500 ml centrifuge pots, 10% glycerol stocks, ~12-20 2 mm gap electroporation cuvettes are one ice and prechilled. 
7. Pre-chill the centrifuge to 4°C.  
8. Pre-warm SOC medium for recovery in a water bath to 37°C.
9. When cells have reached the required OD, immediately split the culture between 2x 500 ml sterile centrifuge pots and place culture on ice.
This will equate to ~340 ml per pot
10. Incubate on ice for 30 mins
11. Spin at 5000 g for 15 mins at 4°C using the JLA-15,000 rotor (harvesting)
Make sure pots have been balanced to within 0.05 g and make sure all 6 positions of the rotor are filled.
12. GENTLY re-suspend each pellet in 25 ml 10% glycerol using a strippette, make up to 250 ml with 10% ice cold glycerol (approx. half of the pot or initial culture level)
13. First wash: Spin at 5000 g for 15 mins at 4°C using the JLA-15,000 rotor 
14. GENTLY re-suspend each pellet in 25 ml 10% glycerol using a strippette, combine the three samples into one pot and make up to 250 ml with 10% ice cold glycerol solution 
15. Second wash: Spin at 5000 g for 15 mins at 4°C using the JLA-15,000 rotor 
16. GENTLY re-suspend both pellets in 25 ml 10% glycerol using a strippette, combine the two samples into one pot and make up to 50 ml with 10% ice cold glycerol solution
17. Third wash: Spin at 3900 rpm for 15 mins at 4°C using the benchtop centrifuge. 
18. GENTLY re-suspend one pellet in 1 ml of 10% ice-cold glycerol. This will result in ~1.2 ml dense cell culture in 10% glycerol.
19. Add 0.2 ul of EZ-Tn5™ [KAN-2] transposon for 100 ul of every competent cells.
20.  Aliquot 100 µl competent cells and transposon mixture to prechilled 0.2 mm cuvettes 
21. Electroplate at 22 kV
This may vary for your strain, e.g. for Salmonella electroporate at 25 kV
22. Immediately add 900 ul pre-warmed SOC medium
23. Transfer the all the electroporated cells to 50 ml (~12 ml) flask and recover in shaking incubator for 2 hours at 37 °C
24. Dilute recovered cells by adding 7 ml SOC directly into the flask making it up to 20 ml
25. Select the cells on selective medium with appropriate antibiotic.

Day 3
26. Collect the transformants and transfer the re-suspension to a 50 ml falcon tube without clumps.

Preparation of transposon junctions for sequencing
 

Protocol for use with NEBNext Ultra DNA Library Prep Kit for Illumina (E7370), refer to the user manual here: 
https://www.neb.com/-/media/catalog/datacards-or-manuals/manuale7370.pdf
Starting Material: (5 ng–1 μg) of fragmented DNA.  We use the max amount of 1 μg, as transposon-junctions are underrepresented within the fragmented genomic DNA.
 

1.1 NEBNext End Prep kit Ultra I (This step is same as the manufacturer’s instructions from the kit)
This step is to repair the ends of the DNA fragments following DNA shearing.
1. Mix the following components in a 0.5 ml sterile nuclease-free PCR tube:
   3.0 µl  End Prep Enzyme Mix - do not vortex
   6.5 µl  End Repair Reaction Buffer (10X)
   50 µl Fragmented DNA
   5.5 µl Water (make up to 65 µl, if <50 µl of sample is recovered from shearing step)
   ----------------------------------------------- 
   65 µl Total volume
   Keep all reagents on ice, and enzymes on cold block.

2. Mix by pipetting.
3. Place in a thermocycler, with the heated lid on, and run the following program:
   30 mins @ 20°C
   30 mins @ 65°C
   Hold at 4°C
 

1.2 Adaptor Ligation (This step is same as the manufacturer’s instructions from the kit)

1. Add the following components directly to the End Prep reaction mixture and mix well:
   15 μl  Blunt/TA Ligase Master Mix
   2.5 µl NEBNext Adaptor for Illumina 
   1 µl Ligation enhancer
   ----------------------------------------------- 
   83.5 µl Total volume

 *The NEBNext adaptor is provided in NEBNext Singleplex (NEB #E7350) or Multiplex (NEB #E7335, #E7500, #E7710, #E7730, #E7600, #E6609) Oligos for Illumina.

2. Mix by pipetting followed by a quick spin to collect all liquid from the sides of the tube.
3. Incubate at 20°C for 15 mins in a thermal cycler.
4. Add 3 μl of USER™ enzyme to the ligation mixture from Step 3. 
Note: This step is only required for use with NEBNext Adaptors. USER enzyme can be found in the NEBNext Singleplex (NEB #E7350) or Multiplex (NEB #E7335, #E7500, #E7710, #E7730, #E7600and #E6609) Oligos for Illumina.
5. Mix well and incubate at 37°C for 15 minutes.
 

1.3 Size Selection or Cleanup of Adaptor-ligated DNA (This step is based on the manufacturer’s instructions from the kit)
The following size selection protocol is for libraries with 200 bp inserts only. For libraries with different size fragment inserts, refer to Table 1.1 in E7370 User Manual for the appropriate volume of beads to be added. The size selection protocol is based on a starting volume of 100 μl.
1. Vortex room-temperature AMPure XP Beads thoroughly to resuspend.
2. Transfer the sample to 1.5 ml LoBind Eppendorf tubes
3. Add 13.5 μl dH2O to the ligation reaction for a 100 μl total volume.
If you have less than 86.5 µl of sample, add water to make the reaction volume up to 100 µl before adding beads
4. Add 55 μl of resuspended AMPure XP Beads to the 100 μl ligation reaction, Mix well by pipetting up and down at least 10 times. MAKE SURE you change tips between samples
The ratio of beads to sample is critical, make sure no beads are on the exterioir of the tip, and that there are no bubbles in the beads
5. Incubate for 5 minutes at room temperature.
6. Place the tube on an appropriate magnetic stand to separate the beads from the supernatant. After the solution is clear (2-5 minutes), carefully transfer the supernatant containing your DNA to a new 1.5 ml Eppendorf (Caution: do not discard the supernatant). Discard the beads that contain the unwanted large fragments.
If beads are disturbed, leave 2 minutes to re-settle on the magnet then try again.
7. Add 25 μl resuspended AMPure XP Beads to the supernatant, mix well by pipetting and incubate for 5 minutes at room temperature.
8. Place the tube on an appropriate magnetic stand to separate the beads from the supernatant. After the solution is clear (about 5 minutes), carefully remove and discard the supernatant that contains unwanted DNA. Be careful not to disturb the beads that contain the desired DNA targets (Caution: do not discard beads).
If beads are disturbed, leave 2-5 minutes to re-settle on the magnet then try again.
9. Keep tubes on the magnetic stand. Add 200 μl of 80% freshly prepared ethanol to each tube. Incubate at room temperature for 30 seconds, and then carefully remove and discard the ethanol.
In practice, if washing multiple tubes, discard the ethanol from the first as soon as it has been added to the last.
10. Repeat the ethanol wash once more.
11. Remove residual ethanol from the bottom of the tubes, be careful not to disturb beads.
12. Air dry the beads for up to 5 minutes with the lids open. Caution: Do not overdry the beads. This may result in lower recovery of DNA target.
13. Elute the DNA target from the beads in 17 μl Buffer EB Qiagen. Mix well by pipetting up and down. Incubate for 2 minutes at room temperature (off the magnetic stand). 
14. Place tubes on a magnetic stand. After the solution is clear (about 5 minutes), transfer 15 μl to a new PCR tube for amplification.
If you are trouble-shooting, take 16 µl at this step, and use spare 1 µl for checks e.g. qubit or nanodrop or bioanalyzer
15. Proceed to PCR Amplification in Section 1.4.
 

1.4 PCR Amplification – Amplification of the transposon junction
1. Mix the following components in a sterile nuclease-free tube:

15 μl  Adaptor Ligated DNA Fragments (our samples)
25 μl  Q5 polymerase
2.5μl  10 µM PCR1-F Primer*
2.5μl  10 µM PCR1-R Primer**
 5 μl   nuclease free water
----------------------------------------------- 
Total volume 50 μl

*This primer is specific for the transposon
**This primer is the same for every library as it is specific for the adapter.

2. PCR cycling conditions (~20 min)

3. Proceed to Cleanup of PCR Amplification Section 1.5.

1.5 Cleanup of PCR Amplification (This step is based on the manufacturer’s instructions from the kit)
1. Vortex AMPure XP Beads to resuspend, allow to come to RT.
2. Transfer PCR reaction to 1.5 ml Eppendorf tube
3. Add 45 μl (ratio 0.9:1) of resuspended AMPure XP Beads to the PCR reactions (~ 50 μl). Mix well by pipetting up and down at least 10 times. Change tips between each sample
4. Incubate for 5 minutes at room temperature.
5. Quickly spin the tube and place it on an appropriate magnetic stand to separate beads from supernatant. After the solution is clear (about 5 minutes), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets (Caution do not discard beads).
6. Add 200 μl of freshly prepared 80% ethanol to the tubes while in the magnetic stand. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant.
7. Repeat ethanol wash once.
8. Remove residual ethanol
9. Air dry the beads for up to 5 minutes with the lids open. Caution: Do not overdry the beads. This may result in lower recovery of DNA target.
10. Elute target DNA from beads in 17 μl EB Buffer. Mix well by pipetting up and down at least 10 times, incubate at room temperature for 2 minutes.
11. Place the sample on an appropriate magnetic stand to separate beads from supernatant. After the solution is clear (2-5 minutes), carefully transfer 15 μl supernatant to a new PCR tube.
If you are trouble shooting, take 16 µl at this step, and use spare 1 µl for checks e.g. qubit or nanodrop
 

1.6 Second PCR Amplification with nested primers
15 μl  Adaptor Ligated DNA Fragments (sample)
25 μl  Q5 polymerase
2.5μl  10 µM inline index custom forward primer (In-house designed primers)
2.5µl   10 µM Illumina index primer (E7335 & E7500) (use same one per condition)
5 μl   Nuclease free water
----------------------------------------------- 
Total volume 50 μl
 

PCR cycling conditions (~40 min)

1.7 Cleanup of PCR Amplification – same as step 1.5 above
1. NB. Final elution in a volume of 33 μL EB, transfer 32 μL to a nuclease free labelled Eppendorf.
2. Prepped libraries can be safely stored at -20°C
 

DATA analysis

Samples were sequenced using an Illumina MiSeq with 150-cycle v3 cartridges. Data were demultiplexed using the Fastx barcode splitter to remove the inline barcode unique to each sample. The transposon was matched and trimmed allowing for 4-bp mismatch, and surviving reads were mapped to the BW25113 reference genome using bwa mem (GenBank accession number CP009273.1). Publicly available TraDIS data analysis pipeline BioTraDIS was used to analyse the demultiplexed data after trimming away low-quality reads. The BioTraDIS analysis package (version 1.4.5) was used to calculate the log fold change in read depth between each gene in the control and dpaA transposon libraries.