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10 µM QP1 primer 1.5 300 nM
10 µM QP2 primer 1.5 300 nM
Nuclease-free water up to 20 —
 cr ItIcal 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.
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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 °C
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 (ml) Final amount
Phusion HF PCR master mix 25 1×
with HF buffer (2×)
10 µM QP1 primer 2.5 500 nM
10 µM QP2 primer 2.5 500 nM
652 | VOL.10 NO.5 | 2015 | nature protocols
## Page 9
protocol
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.
10. Transfer the supernatant to new nuclease-free tubes without disturbing the beads.
 cr ItIcal step KAPA HiFi HotStart ReadyMix can be used as an alternative to Phusion HF PCR master mix with HF buffer,
if available.
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29| Add 30 µl of the second-round PCR mixture from Step 28 to the tube from Step 27 so that the total volume of the PCR
mixture is 50 µl: 20 µl of eluted DNA and 30 µl of second-round PCR mixture. Mix well by vortexing and centrifuge the
mixture again at 9,000g for 1 min at 4 °C.
30| Perform another 25 PCR cycles with the following thermocycler program:
cycle number Denature anneal extend Hold
1 98 °C, 2 min
2–26 98 °C, 10 s 60 °C, 30 s 72 °C, 1 min
27 72 °C, 5 min
28 4 °C
31| After the PCR cycles, centrifuge the tubes at 9,000g for 1 min at 4 °C. Next, purify the PCRs once using 1:1-fold AMPure
XP beads, as described in Box 1; finally, elute in 20 µl of nuclease-free water.
size selection of the amplified Dna fragments ● tIMInG 9–10 h
32| Prepare the 12% (wt/vol) native polyacrylamide TBE gel (Reagent Setup) using the Bio-Rad electrophoresis system.
 cr ItIcal step As more primer dimers are generated in the scRRBS protocol than in regular RRBS protocols, we recommend