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13| Prepare the end-repair/dA-tailing mixture, as described in the following table. Mix well by vortexing, and then centrifuge the mixture at 9,000g for 1 min at 4 °C. |
Component Amount per reaction (μl) Final amount |
5 U/μl Klenow Fragment, exo- 1 5 U |
10× Tango buffer 0.2 1x |
End-repair dNTP mix 0.8 40 μM dATP, 4 μM dCTP, 4 μM dGTP |
14| Add 2 μl of end-repair/dA-tailing mixture from Step 13 to each tube from Step 12 so that the total volume of each sample is 20 μl: 18 μl of MspI digested reaction and 2 μl of end-repair/dA-tailing mixture. Mix it well by vortexing the tubes, and centrifuge the mixture at 9,000g for 1 min at 4 °C. |
15| Incubate the reaction at 37 °C for 40 min, and then at 75 °C for 15 min to inactivate the enzyme. Centrifuge the tubes at 9,000g for 1 min at 4 °C, and place the tubes on ice immediately. |
## Adapter ligation TIMING 9-10 h |
16| Prepare the adapter ligation mixture as described in the following table. Mix it well by gentle inversion, and centrifuge the mixture at 9,000g for 1 min at 4 °C. |
Component Amount per reaction (μl) Final amount |
30 Weiss U/μl T4 DNA ligase 1 30 Weiss U |
10× Tango buffer 0.5 1x |
100 mM ATP 0.25 1 mM |
1:20 diluted adapter 1 |
Nuclease-free water up to 5 |
▲ CRITICAL STEP Do not vortex the mixture, as vigorous vortexing may damage the Y-shaped adapters. |
17|Add 5 μl of ligation mixture from Step 16 to each tube from Step 15 so that the total volume of each sample is 25 μl: 20 μl of end-repaired and dA-tailed reaction and 5 μl of ligation mixture. Mix the reaction well by gentle inversion, and centrifuge the mixture at 9,000g for 1 min at 4 °C. |
18| Incubate the reaction at 16 °C for 30 min, followed by 4 °C overnight (at least 8 h). |
19| Heat-inactivate the enzyme reaction at 65 °C for 20 min. Next, centrifuge the tubes at 9,000g for 1 min at 4 °C and immediately place the tubes on ice. |
PAUSE POINT The reaction may be stored at -80 °C for no longer than 3 months if necessary. |
## Bisulfite conversion TIMING 3-4 h |
20| Use all of the 25-μl ligation mixture from Step 19 to perform the bisulfite treatment. We use the MethylCode bisulfite conversion kit, according to the manufacturer's instructions, as summarized in Steps 20-23: add 125 μl of well-dissolved CT conversion reagent (Reagent Setup) to the 25-μl ligation mixture, mix wel... |
21| Perform the bisulfite conversion under the following conditions on the thermocycler: first at 98 °C for 10 min, then at |
64 °C for 2.5 h and finally at 4 °C for temporary storage. |
22| Purify the converted DNA by using the MethylCode bisulfite conversion kit according to the manufacturer's instructions. |
1 μl of tRNA (10 ng/μl) should be added to the supplied DNA binding buffer to act as a protective carrier. |
23| Elute the converted DNA with 30 μl of preheated (50 °C) elution buffer (included in the MethylCode bisulfite conversion kit) according to the kit instructions. |
PAUSE POINT The eluted DNA may be stored at -80°C for at least 6 months. |
## First-round PCR enrichment TIMING 3-4 h |
24| Prepare the first-round PCR mixture as described in the following table. Mix it well by vortexing, and centrifuge the mixture at 9,000g for 1 min at 4 °C. |
Component Amount per reaction (μl) Final amount |
10× PCR Pfu Cx Buffer 5 1× |
2.5 U/μl Pfu Turbo Cx hotstart DNA polymerase 0.4 1U |
dNTP mix (10 mM each) 1 200 μM (each) |
10 μM QP1 primer 1.5 300 nM |
10 μM QP2 primer 1.5 300 nM |
Nuclease-free water up to 20 |
▲ CRITICAL STEP PfuTurbo Cx hotstart DNA polymerase must be used in this step because this enzyme is resistant to uracil stalling, thus ensuring that both methylated and unmethylated DNA fragments will be amplified with similar efficiencies. ? TROUBLESHOOTING |
25| Add 20 μl of the first-round PCR mixture from Step 24 to the tube from Step 23 so that the total volume of the PCR mix is 50 μl: 30 μl of eluted converted DNA and 20 μl of first-round PCR mixture. Mix it well by vortexing, and then centrifuge the mixture at 9,000g for 1 min at 4 °C. |
26| Perform 25 cycles of PCR on the PCR thermocycler and run the following program: |
Cycle number Denature Anneal Extend Hold |
1 95 °C, 2 min |
2-26 95 °C, 20 s 60 °C, 30 s 72 C, 1 min |
27 72 °C, 5 min |
28 4℃ |
27| After the PCR cycles, centrifuge the tubes at 9,000g for 1 min at 4 °C. Next, purify the PCRs twice via 1:1-fold AMPure XP beads, as described in Box 1, and finally elute in 20 μl of nuclease-free water. |
## Second-round PCR enrichment TIMING 3-4 h |
28| Prepare the second-round PCR mixture as described in the following table. Mix well by vortexing, and then centrifuge the mixture at 9,000g for 1 min at 4 °C. |
Component Amount per reaction (μl) Final amount |
Phusion HF PCR master mix with HF buffer (2×) 25 1× |
10 μM QP1 primer 2.5 500 nM |
10 μM QP2 primer 2.5 500 nM |
## Box 1 DNA purification using AMPure XP beads TIMING 0.5-1 h |
1. Remove the AMPure XP bead suspension from storage and vortex the AMPure XP beads until they are well dispersed. Let the bead suspension stand for at least 30 min to bring the beads to room temperature. |
2. Add an equal volume of well-dispersed AMPure XP bead suspension to the reaction tubes (e.g., 50 μl of suspension to 50 μl of the reaction solution) and mix well by repeated pipetting. |
3. Incubate the mixture at room temperature for 15 min, and then place the tubes on the magnetic rack for at least 5 min until the solution becomes clear. |
4. Remove and discard the supernatant carefully to avoid disturbing the beads. |
5. Add 200 μl of freshly prepared 80% (vol/vol) ethanol and invert the tubes several times with a magnetic rack; carefully discard the supernatant. |
6. Repeat the wash step once more as described in Step 5. |
7. Let the tubes stand at room temperature for at least 10 min with the lids open until the beads are dry. |
8. Resuspend the dried beads with an appropriate volume (usually 20-50 μl) of nuclease-free water, mix well by repeated pipetting and incubate the mixture at room temperature for at least 2 min. |
9. Place the resuspended solution on the magnetic rack again, and then let the tubes stand for at least 5 min until the solution becomes clear. |
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