TITLE: stLFR library construction
AUTHORS: Chunhua Li, Xianwei Yang, Libin Shao, Rui Zhang, QunLiu, Mengqi Zhang, Shanshan Liu, Shanshan Pan, Weizhen Xue, Congyan Wang, Chunyan Mao, He Zhang, Guangyi Fan

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used to clarity the process of stLFR library preparation for Bicolor Angelfish (Centropyge bicolor) genome.</div></div>

[STEPS]
?. Sample preparationRemove the long-fragment gDNA from  and store on ice.
4 °C
?. Transposon Insertion
?. Transfer  long-fragment gDNA gently into a 0.2ml PCR tube. Without mixing, add Nuclease Free Water to a total volume of . Collect and dispense long fragment DNA slowly (the process should take >) each time when pipetting.
10 ng
36.8 µl
?. Prepare the stLFR_SamBarTIE and TE Buffer. Dilute the stLFR_SamBarTIE 16-fold with TE Buffer. Add  TE Buffer into a new 0.2 mL PCR tube and then transfer  stLFR_SamBarTIE to the tube. Vortex intermittently for 4 times (2s each) to mix. Label it as “4× dilution stLFR_SamBarTIE”.
6 µl
2 µl
?. Add  TE Buffer into a new 0.2 mL PCR tube and transfer  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.
18 µl
6 µl
?. 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  Transposon Insertion Reaction Mix to the DNA sample from step 2.1(total volume is ). 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.
13.2 µl
50 µl
?. Store the tubes on ice after the transposon insertion step has finished.
?. Pipette  TE buffer to a new 0.2 mL tube then transfer  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.
42.5 µl
7.5 µl
?. Capture
?. Vortex Capture Beads to mix thoroughly before use. Pipette  Capture Beads per sample to the tube.
30 µl
?. Place the tube on a magnetic separation rack. Once the liquid is clear, carefully collect and discard the supernatant.
?. Pipette  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.
50 µl
?. 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  Capture Buffer per sample to resuspend the Capture Beads.
50 µl
?. Transfer  Capture Beads from step 3.4 to the sample tube from step 2.7 and mix thoroughly by inverting gently at least 10 times.
50 µl
?. 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  of incubation at , switch the temperature of the incubator to . Open the door of the incubator to accelerate cooling, then close the door of the incubator and start the  countdown once the temperature drops to .
60 °C
45 °C
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  Ligation Reaction 1 Mix to the  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.
30 µl
100 µl
?. Perform Ligation Reaction 1 with the incubation in  (20°C to 25°C) . After incubation, centrifuge the sample and place it on the magnetic separation rack. Carefully remove and discard the supernatant once the liquid is clear.
4 Room temperature
?. Pipette  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.
180 µl
?. Digestion Reaction 1
?. Transfer  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).
100 µl
?. Place the sample tube on the rotator and turn it on. Perform the Digestion Reaction 1 incubation in . Remove the sample tube from the incubator at the end of the reaction. Immediately add TIS Buffer.
37 °C
?. Termination Reaction
?. Remove the sample from , centrifuge briefly, and store at room temperature. Immediately add  TIS Buffer to each sample from step 5.2.
37 °C
11 µl
?. 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  and place on the rotator. Start the rotator and perform the incubation in  (20°C to 25°C) 10 minutes.
37 Room temperature
?. 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  Wash Buffer II into the sample tube. Mix the beads by vortexing for . Centrifuge briefly and place the tube back onto the magnetic rack for . Carefully remove and discard the supernatant once the liquid becomes clear. Repeat this step twice.
150 µl
?. Pre-Ligation Reaction 2
?. Pipette  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  and place on the rotator stored in the  incubator. Perform the incubation in .
24 µl
37 °C
37 °C
?. When the Pre-Ligation 2 Reaction is complete, remove the product from incubator immediately and centrifuge briefly. Keep it at  and proceed to next step.
37 Room temperature
?. Ligation Reaction 2
?. Pipette all  Ligation Reaction 2 mix into the sample tube for a total volume of . Mix the sample tube by vortexing for . Centrifuge the tube for  to make sure beads are fully resuspended.
76 µl
100 µl
?. Place the tube on rotator and perform the incubation in  (20°C to 25°C) .
37 Room temperature
?. After incubation, centrifuge the sample and pipette  Wash Buffer II into the sample tube. Place the sample tube on the magnetic separation rack for. Carefully remove and discard the supernatant when the liquid becomes clear.
80 µl
?. Keep the sample tube on the magnetic separation rack and pipette  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.
180 µl
?. PCR
?. Pipette  of the PCR mix into sample tube. Use the pipet to mix the beads until fully resuspend. Transfer  of the sample to a different 0.2 mL tube.
150 µl
75 µl
?. 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  and equilibrate to  for at least  before use. Vortex at full speed for  to ensure the beads are completely resuspended.
4 °C
4 Room temperature
?. 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  for .
4 Room temperature
?. Centrifuge the tube and place on the magnetic separation rack. Wait for  or until the solution is clear. Discard the supernatant.
75 µl
?. Keep the sample tube on the magnetic separation rack and transfer  80% (v/v) ethanol into the tube. Let stand for . Carefully remove and discard the supernatant. Repeat this step once more.
500 µl
?. 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  TE Buffer for elution. Vortex for  to resuspend the beads, then briefly centrifuge.
33 µl
?. Incubate the sample at  for .
4 Room temperature
?. Place the sample tube on the magnetic separation rack and wait for  or until the supernatant is clear. Transfer  of supernatant from the sample tube to a new 1.5 mL EP tube. Do not disturb or pipette the beads.
31 µl