STLFR library construction for snake genomes

Sample preparation

Remove the long-fragment gDNA from 4°C and store on ice.

Transposon Insertion

Transfer 10ng long-fragment gDNA gently into a 0.2ml PCR tube. Without mixing, add Nuclease Free Water to a total volume of 36.8µL. Collect and dispense long fragment DNA slowly (the process should take >0h 0m 10s) each time when pipetting.

Prepare the stLFR_SamBarTIE and TE Buffer. Dilute the stLFR_SamBarTIE 16-fold with TE Buffer. Add 6µL TE Buffer into a new 0.2 mL PCR tube and then transfer 2µL stLFR_SamBarTIE to the tube. Vortex intermittently for 4 times (2s each) to mix. Label it as “4× dilution stLFR_SamBarTIE”.

Add 18µL TE Buffer into a new 0.2 mL PCR tube and transfer 6µL of the 4× dilution stLFR_SamBarTIE into the tube. Vortex intermittently for 4 times (2s each) to mix the tube. Label it as “stLFR_SamBarTIE (Working Mix).” stLFR_SamBarTIE (Working Mix) can be used for 12 reactions.

According to the requirements of the sample sequencing pooling strategy, mix the stLFR_SamBarTIE Working Mix.

Prepare the Transposon Insertion Reaction Mix on ice as shown. Vortex intermittently for 4 times (2s each) to mix.

Transfer 13.2µL Transposon Insertion Reaction Mix to the DNA sample from step 2.1(total volume is 50µL). Mix by very gently pipetting 10 times using wide-bore tip. Briefly centrifuge the tube. Transfer the tube to the thermocycler and start the Transposon Insertion Reaction Program.

Store the tubes on ice after the transposon insertion step has finished.

Pipette 42.5µL TE buffer to a new 0.2 mL tube then transfer 7.5µL transposon-inserted product (from step 2.6) to this new 0.2 mL tube. Mix by inverting the tube very gently and collect liquid to the bottom of the tube by briefly centrifuging (1 second) on a microcentrifuge. Label this new tube as the sample tube.

Capture

Vortex Capture Beads to mix thoroughly before use. Pipette 30µL Capture Beads per sample to the tube.

Place the tube on a magnetic separation rack. Once the liquid is clear, carefully collect and discard the supernatant.

Pipette 50µL Wash Buffer I per sample into the 0.2 mL PCR tube or 1.5 mL EP tube. Ensure that Wash Buffer I can cover all of the Capture Beads.

Rotate the tube 180 degrees within the rack such that the beads are forced to pass through the Wash Buffer I. Repeat the tube rotation once. Carefully remove and discard the supernatant once the liquid in the tube is clear. Pipette 50µL Capture Buffer per sample to resuspend the Capture Beads.

Transfer 50µL Capture Beads from step 3.4 to the sample tube from step 2.7 and mix thoroughly by inverting gently at least 10 times.

Centrifuge the product from step 3.5 for 1 second and place on the rotator in the incubator. Immediately start rotating the sample. In this experiment, After the first 0h 10m 0s of incubation at 60°C, switch the temperature of the incubator to 45°C. Open the door of the incubator to accelerate cooling, then close the door of the incubator and start the 0h 50m 0s countdown once the temperature drops to 48°C.

Ligation Reaction 1

Centrifuge the product from step 3.6 and allow the product to cool to room temperature.

Ensure the product has cooled to room temperature, then transfer 30µL Ligation Reaction 1 Mix to the 100µL sample. Mix by inverting the tubes gently at least 10 times then briefly centrifuge (1s). Place the sample tube on the rotator and turn it on.

Perform Ligation Reaction 1 with the incubation in 4Room temperature (20°C to 25°C) 0h 10m 0s. After incubation, centrifuge the sample and place it on the magnetic separation rack. Carefully remove and discard the supernatant once the liquid is clear.

Pipette 180µL of Wash Buffer II into the sample tube. Rotate the tube 180 degrees while on the magnetic separation rack to let the beads move through the Wash Buffer II. Repeat the tube rotation once. Carefully remove and discard the supernatant once the liquid is clear. Keep the Capture Beads in the Wash Buffer II until the Digestion Reaction Mix 1 is ready.

Digestion Reaction 1

Transfer 100µL Digestion Reaction 1 Mix to the sample tube from step 4.4. Mix by inverting the tube gently at least 10 times followed by an instantaneous centrifugation (1s).

Place the sample tube on the rotator and turn it on. Perform the Digestion Reaction 1 incubation in 37°C 0h 10m 0s. Remove the sample tube from the incubator at the end of the reaction. Immediately add TIS Buffer.

Termination Reaction

Remove the sample from 37°C, centrifuge briefly, and store at room temperature. Immediately add 11µL TIS Buffer to each sample from step 5.2.

Ensure the sample tube is sealed tightly. Mix the sample tube by vortexing at medium speed for 3 to 5 seconds to make sure the beads are fully resuspended. Centrifuge the tube for 0h 0m 1s and place on the rotator. Start the rotator and perform the incubation in 37Room temperature (20°C to 25°C) 10 minutes.

After incubation, centrifuge the sample and place on the magnetic separation rack. Carefully remove and discard the supernatant once the liquid is clear. Keep tube on the magnetic separation rack and pipette 150µL Wash Buffer II into the sample tube. Mix the beads by vortexing for 0h 0m 5s. Centrifuge briefly and place the tube back onto the magnetic rack for 0h 2m 0s. Carefully remove and discard the supernatant once the liquid becomes clear. Repeat this step twice.

Pre-Ligation Reaction 2

Pipette 24µL Pre-Ligation 2 Reaction mix into the sample tube from step 6.3. Mix the sample tube by vortexing for 3-5 seconds to ensure the beads are fully resuspended. Centrifuge the tube for 0h 0m 1s and place on the rotator stored in the 37°C incubator. Perform the incubation in 37°C 0h 10m 0s.

When the Pre-Ligation 2 Reaction is complete, remove the product from incubator immediately and centrifuge briefly. Keep it at 37Room temperature and proceed to next step.

Ligation Reaction 2

Pipette all 76µL Ligation Reaction 2 mix into the sample tube for a total volume of 100µL. Mix the sample tube by vortexing for 0h 0m 10s. Centrifuge the tube for 0h 0m 1s to make sure beads are fully resuspended.

Place the tube on rotator and perform the incubation at 37Room temperature (20°C to 25°C) 2h 0m 0s.

After incubation, centrifuge the sample and pipette 80µL Wash Buffer II into the sample tube. Place the sample tube on the magnetic separation rack for0h 2m 0s. Carefully remove and discard the supernatant when the liquid becomes clear.

Keep the sample tube on the magnetic separation rack and pipette 180µL Wash Buffer II into the sample tube. Rotate the tube 180 degrees within the magnetic separation rack to allow the beads to move through Wash Buffer II. Repeat once. Carefully remove and discard the supernatant when the solution becomes clear. Make sure Wash Buffer II is completely removed.

PCR

Pipette 150µL of the PCR mix into sample tube. Use the pipet to mix the beads until fully resuspend. Transfer 75µL of the sample to a different 0.2 mL tube.

Place all samples on the thermocycler. Make sure the beads are fully resuspended before starting.

Centrifuge the sample and place on the magnetic separation rack. Transfer all the supernatant of the two PCR tube from the same sample into a new 1.5 mL EP tube and mix together.

After confirming complete recovery of the supernatant, discard the original PCR tube.

PCR Product Purification

Remove the DNA Clean beads from 4°C and equilibrate to 4Room temperature for at least 0h 30m 0s before use. Vortex at full speed for 0h 0m 10s to ensure the beads are completely resuspended.

Measure the volume of the PCR product from step 9.3. Add 0.7-fold DNA Clean beads to the PCR product. Vortex the tube to mix the beads with the sample.

Incubate the sample at 4Room temperature for 0h 10m 0s.

75µL Centrifuge the tube and place on the magnetic separation rack. Wait for 0h 2m 0s or until the solution is clear. Discard the supernatant.

Keep the sample tube on the magnetic separation rack and transfer 500µL 80% (v/v) ethanol into the tube. Let stand for 0h 0m 30s. Carefully remove and discard the supernatant. Repeat this step once more.

Keep the sample tube on the magnetic separation rack, open the cap of tube and air-dry the beads for 3 to 5 minutes until no wetness is observed (the surface of the beads will dim). Do not over dry the beads as this will significantly decrease the elution efficiency (cracks can be observed on pellet).

Remove the sample tube from the magnet and add 33µL TE Buffer for elution. Vortex for 0h 0m 3s to resuspend the beads, then briefly centrifuge.

Incubate the sample at 4Room temperature for 0h 5m 0s.

Place the sample tube on the magnetic separation rack and wait for 0h 2m 0s or until the supernatant is clear. Transfer 31µL of supernatant from the sample tube to a new 1.5 mL EP tube. Do not disturb or pipette the beads.