Generating Illumina Sequencing Libraries for transposons created in A. baylyi ADP1 with the pBT20 vector.

This protocol prepares Illumina sequencing libraries from Acinetobacter baylyi ADP1 mutagenized with the pBT20 conjugation vector. It is based on the tn-seq approach used by Eli Powell in the Moran lab in Snodgrassella alvi. Note: low retention eppindorf tubes and sterile filter tips should be used to avoid contaminating reagents and cross contaminating samples.

Uncommon lab materials needed before starting

• Covaris tubes for shearing
• dCTP
• ddCTP
• Promega rTdT with 5X reaction buffer
• AMPure beads
• KOD Hifi DNA polymerase with buffers
• Steptavidin-coupled M-280 Dynabeads
• ADP1 Tn-seq primer set (see below)

ADP1 Tn-seq Library Prep Protocol

Preparation of sheared gDNA of ADP1 Tn-libraries

• Isolate 8 µg of gDNA using the Invitrogen PureLink Mini Genomic DNA prep kit from each population to be sequenced. If working from frozen cell pellet, use 15 /µL of pelleted cells per prep. Multiple preps per population may be required to get 8 µg. Use the kit's elution buffer for the elutions. Quantify the DNA concentration with the Qubit.
• Transfer the gDNA in a 50 µL volume to a Covaris tube. If the DNA concentration is too low, dehydrate the gDNA in the speedvac and resuspend it in 50 µL dH2O.
• Go to the Covaris machine in the core (making sure it is on and has been degassing > 30 minutes) and shear the sample with the Barrick Lab 300bp protocol.
• Transfer the DNA into a fresh Eppindorf tube.

Cleaning of DNA with AMPure beads

This protocol is to be used with noted modifications throughout this procedure.

• Pull tube of beads out of the 4°C fridge and let rest on the bench until they reach room temperature.
• In a low retention eppindorf tube, combine DNA and beads in the appropriate ratio. For this step, a 1.2 bead:DNA ratio is required. To do this, combine 50 µL of sheared DNA and 60 µL of beads and mix by pipetting. Let the tube incubate at room temperature for 10 minutes.
• Plate the tube on a magnet stand. Let sit until the beads are pulled out of solution. Remove and discard the liquid without disturbing the beads.
• With the tube still on the magnet, add 200 µL of 80% (v/v) ethanol (made fresh that day). Let sit ~ 5 sec and remove via pipetting. Repeat this step once.
• After pipetting off all of the ethanol, let the beads air dry such that they are no longer wet but not to the point of the beads cracking. This will be < 5 minutes.
• Resuspend the beads in the desired elution volume. For this particular part of the protocol, elute the samples in 25 µL dH2O. After the beads are resuspended, put the tube back on the magnet to remove the beads. Transfer the bead-free liquid to another eppinforf tube. Quantify the concentration of DNA with the Qubit.

Terminal deoxynuucleotidyl transferase (TdT) tailing reaction

This step adds poly-C tails to the sheared fragments.

Setup the following reaction for each library:

• 2.5 µg cleaned and sheared gDNA in 36.125 µL dH2O
• 10 µL 5X TdT reaction buffer
• 2.375 µL 10mM dCTP
• 0.25 µL 5mM ddCTP
• 1.2 µL rTdT

Incubate the reaction at 37°C for 1 hour.

Size Selection

This step ensures that very large fragments and very short fragments are removed before PCR steps.

• Transfer 48 µL of the poly-C reaction to a new tube and add 24 µL of AMPure beads, mix via pippeting, and incubate at room temperature for 10 minutes.
• Separate the beads with the magnet and transfer the supernatant to a new tube. (The high molecular weight DNA is precipitated on the beads and will be discarded).
• To the supernatant tube add 33.6 µL more beads, mix via pippeting, and incubate at room temperature for 10 minutes.
• Following the normal bead washing procedure and elute the DNA in 25 µL dH2O. Qubit to quantify.

PCR #1

This first round PCR adds the biotin tag, amplifying from inside the transposon, and the first part of the Illumina adapters, amplyfing from the poly-C tail. All reagents are from the KOD polymerase kit. Set one reaction per library.

• 5 µL KOD buffer 1
• 3 µL MgCl2
• 5 µL 2mM dNTPs
• 0.75 µL 20 µM FW primer (see primer list)
• 2.25 µL 20 µM RV primer (see primer list)
• 0.8 µL KOD polymerase
• 250 ng poly-C tailed size selected DNA
• dH2O to 50 µL.

PCR Protocol to use:

• 2 min 95°C
• 15 sec 95°C
• 30 sec 65°C
• 30 sec 72°C
• Go to step 2 19X
• hold at 16°C

After PCR, purify the 50 µL reactions with 60 µL of beads. Elute in 50 µL dH2O.

Binding of library to streptavidin beads

This step binds the PCR products to the paramagnetic beads to enrich them for the second round of PCR.

• Resuspend the beads (Streptatividin coupled paramagnetic beads M280) in the bottle via vortexing and aliquiot 32 µL into a fresh eppindorf tube for each library.
• Add 1mL 1X B&W buffer and vortex to wash the beads. Spin down briefly to remove liquid from cap top and put on the magnet. Pipet off the liquid and discard. Repeat this wash step fr a total of 3 washes.
• Resuspend the beads in 52 µL 2X B&W buffer.
• Add 50 µL cleaned PCR 1 to the tube. Mix via pipetting.
• Rotate at room temperature for 30 minutes.
• Wash once again with 1 mL B&W buffer.
• Wash twice with 1mL LoTE.
• Resuspend beads in 25 µL LoTE.

Final PCR

Adds remaining Illumina indexes and primer sites.

• 25 µL bead+DNA mixture.
• 5 µL 10X KOD buffer 1
• 5 µL 2mM dNTPs
• 2 µL MgCl2
• 3 µL 10 µM FW primer (see primer list)
• 3 µL 10 µM RV primer (see primer list)
• 6.2 µL dH2O
• 0.8 µL KOD polymerase

PCR Protocol:

• 2 min 95°C
• 20 sec 95°C
• 30 sec 53°C
• 30 sec 72°C
• Go to step 2 19X
• Hold at 16°C

Once complete, transfer the reaction to an Eppindorf and remove the beads with a magnet. Clean up with 60 µL AMPure beads and elute in 25 µL dH2O. Qubit and submit a sample to the core for bioanayzer analysis. This can be done on a pooled library sample.

-- Main.BrianRenda - 07 Apr 2016