1.DNA tagmentation with Tn5
a. Prepare a mix consisting of 200 µL 5xTAPS buffer, 50 µg of genomic DNA, and the appropriate volume of 22 µM Tn5 (200 µL for HEK293T). Add water to reach a total volume of 1000 µL. Incubate the mixture on a bath shaker at 55°C and 1000 rpm for 7 minutes. Ensure that the resulting DNA size ranges from 0.5-1.5kb.
b. Purify DNA according to the Tiangen Universal DNA Purification kit manual, using six reactions per sample. Elute with 70 µl water per reaction and combine. Perform LAM-PCR.
2.LAM-PCR
c. Prepare 16 PCR reactions for each sample: 10 µL 5xFastpfu buffer, 1 µL 2.5 mM dNTPs,1 µL 1 µM bio-primer, 0.5 µL Fastpfu, 25 µL tagmented DNA, 12.5 µL water.
Follow the below PCR program: Heat the mixture to 95°C for 5 minutes. Perform 20 cycles of the following steps: Denature at 95°C for 30 seconds, anneal at 58°C for 30 seconds, and extend at 72°C for 1 minute and 30 seconds. Then, extend for 72°C for 5 minutes and maintain at 10°C indefinitely.
d. Add 60 µL PCR beads (in room temperature) into each PCR tubes, mix gently, and allow the tubes to incubate at room temperature for 5 minutes.
e. Return PCR tube to the 96-well magnet stand and allow the beads to sit for 2 minutes.
f. Discard the supernatant and wash the beads with 200 µL of 70% ethanol twice.
g. Dry the beads for 3-5 min after removing the ethanol.
h. Resuspend the beads in 10 µL Tris-HCl (10 mM, pH7.4) for each tube, mix the beads with a pipette at least 10 times and incubate at room temperature for 2 min.
i. Place the beads against a magnet stand for 5 min and collect the supernatant.
3.C1 beads enrichment
j. Add 15 µL of Dynabeads MyOne C1 for each sample in a 1.5 mL tube; wash the beads with 600 µL of 1x B&W buffer three times.
k. Collect beads in the magnet and add 40 µL 5M NaCl and 2 µL EDTA for each sample to resuspend beads.
l. Mix resuspended beads with supernatant from step I and rotate at room temperature for 2 hours.
4.On-beads PCR
m. Prepare two PCR reactions for each sample: 5 µL 10x EasyTaq PCR buffer, 4 µL 2.5 mM dNTPs, 4 µL 10 µM extra primer, 4 µL 10 µM index primer, 0.5 µL EasyTaq polymerase, 25 µL DNA-beads complex, 11.5 µL water.
PCR program: 95 oC for 5 minutes; [95 oC for 1 minute, 58 oC for 30 seconds, 72 oC for 1 minute] (10 cycles); 72 oC for 5 minutes; 10 oC forever.
n. Use a 96-well small magnet to capture the beads and transfer the supernatant into new PCR tubes.
o. Add 50 µL PCR beads (in room temperature) into each PCR tubes, mix gently and keep the tubes at room temperature for 5 minutes.
p. Repeat steps e-g. Resuspend the beads in 30 µL water for each tube, mix the beads with pipette at least 10 times and keep at room temperature for 2 minutes.
q. Place the beads against the magnet and allow them to stand for 5 minutes. Then, retrieve the supernatant and transfer it to a 1.5 mL tube.
5.Enzyme blocking
r. Prepare an enzyme digestion mix consisting of 10 µL of 10x Cutsmart buffer, 60 µL of supernatant, 30 µL of water, and 5 units of the restriction enzyme. Incubate the mixture at the recommended temperature for one hour.
s. Purify the DNA according to the Tiangen Universal DNA Purification kit manual and elute it in 50 µl of water.
6.Nest PCR
t. Prepare two PCR reactions for each sample: 5 µL 10x EasyTaq PCR buffer, 4 µL 2.5 mM dNTPs, 4 µL 10 µM red primer, 4 µL 10 µM index primer, 0.5 µL EasyTaq polymerase, 25 µL elution product from step s and 11.5 µL water.
PCR program: 95 oC for 5 minutes; [95 oC for 1 minute, 58 oC for 30 seconds, 72 oC for 1 minute] (10 cycles); 72 oC for 5 minutes; 10 oC forever.
u. Add 50 µL PCR beads (at room temperature) into each PCR tube, mix gently, and leave the tubes at room temperature for 5 minutes.
v. Repeat steps e-g. Resuspend the beads in 35 µL of water per tube, mix them with a pipette at least 10 times, and incubate at room temperature for 2 minutes. Then place the beads against a magnet stand for 5 minutes and recover the supernatant.
7.Tag PCR
x. Prepare 2 PCR reactions for each sample: 10 µL 5x Fastpfu buffer, 4 µL 2.5 mM dNTPs, 4 µL 10 µM P5 primer, 4 µL 10 µM P7 primer, 0.5 µL Fastpfu polymerase, 33.5 µL supernatant.
PCR program: 95 oC for 3 minutes; [95 oC for 20 seconds, 60 oC for 30 seconds, 72 oC for 1 minute] (10 cycles); 72 oC for 5 minutes; 10 oC forever.
y. Run 2% agarose gel, cut smear between 300-700bp.