sequencing/scg-protocols-v1 · Datasets at Hugging Face

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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.



 CRITICAL STEP KAPA HiFi HotStart ReadyMix can be used as an alternative to Phusion HF PCR master mix with HF buffer,reserved. if available.
rights ? TROUBLESHOOTING
All 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
Inc. mixture again at 9,000g for 1 min at 4 °C.

30\| Perform another 25 PCR cycles with the following thermocycler program:America,
Nature Cycle1 number 98Denature°C, 2 min Anneal Extend Hold

2–26 98 °C, 10 s 60 °C, 30 s 72 °C, 1 min2015
© 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.
 CRITICAL STEP As more primer dimers are generated in the scRRBS protocol than in regular RRBS protocols, we recommend
the use of a gel-based approach to thoroughly remove the primer dimers.

33\| Add 4 µl of 6× DNA loading buffer to the sample, mix it well by vortexing and spin down the mixture briefly.

34\| Pour freshly prepared 1× TBE running buffer into the electrophoresis apparatus, and place the gel cassette into the
apparatus. Then, pull out the combs carefully to avoid destroying the wells.

35\| Load 24 µl of well-mixed samples and 2 µl of low-range DNA ladder into different wells. Run the gels at 150 V for ~1 h,
stopping before the bromophenol blue loading dye runs out of the gel.

36\| After electrophoresis, remove the gel cassette from the electrophoresis apparatus, peel the gel off the glass and stain it
with a 1:10,000 dilution of SYBR Gold nucleic acid gel stain in 1× TBE buffer for 10 min at room temperature in darkness on
a shaker.


nature protocols \| VOL.10 NO.5 \| 2015 \| 653

## Page 10

protocol

37\| Wash the gel twice with 1× TBE buffer to remove the stain dye, and then visualize the gel with a Dark Reader
transilluminator in darkness (Fig. 2a).

38\| Excise the gel slices containing 200–700-bp DNA fragments using a clean disposable blade, and then transfer the gel
slices into 0.5-ml thin-walled PCR tubes.
! CAUTION Handle the blade with appropriate protection.

39\| Recover DNA fragments from the gels using an appropriate method. We have found that we achieve maximum recovery
using the ‘crush and soak’ method (Box 2).

40\| Purify the eluate from Step 39 using the QIAquick PCR purification kit (or any equivalent kit) according to the
manufacturer’s protocol. Elute the DNA from the column using 50 µl of preheated EB buffer (part of the QIAquick PCR
purification kit).

41\| Purify the eluate twice using 1:1-fold AMPure XP beads, as described in Box 1, and finally elute in 20 µl of
nuclease-free water.

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.

 CRITICAL STEP KAPA HiFi HotStart ReadyMix can be used as an alternative to Phusion HF PCR master mix with HF buffer,reserved. if available.

rights ? TROUBLESHOOTING

All 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

Inc. mixture again at 9,000g for 1 min at 4 °C.

30| Perform another 25 PCR cycles with the following thermocycler program:America,

Nature Cycle1 number 98Denature°C, 2 min Anneal Extend Hold

2–26 98 °C, 10 s 60 °C, 30 s 72 °C, 1 min2015

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.

 CRITICAL STEP As more primer dimers are generated in the scRRBS protocol than in regular RRBS protocols, we recommend

the use of a gel-based approach to thoroughly remove the primer dimers.

33| Add 4 µl of 6× DNA loading buffer to the sample, mix it well by vortexing and spin down the mixture briefly.

34| Pour freshly prepared 1× TBE running buffer into the electrophoresis apparatus, and place the gel cassette into the

apparatus. Then, pull out the combs carefully to avoid destroying the wells.

35| Load 24 µl of well-mixed samples and 2 µl of low-range DNA ladder into different wells. Run the gels at 150 V for ~1 h,

stopping before the bromophenol blue loading dye runs out of the gel.

36| After electrophoresis, remove the gel cassette from the electrophoresis apparatus, peel the gel off the glass and stain it

with a 1:10,000 dilution of SYBR Gold nucleic acid gel stain in 1× TBE buffer for 10 min at room temperature in darkness on

a shaker.

nature protocols | VOL.10 NO.5 | 2015 | 653

## Page 10

protocol

37| Wash the gel twice with 1× TBE buffer to remove the stain dye, and then visualize the gel with a Dark Reader

transilluminator in darkness (Fig. 2a).

38| Excise the gel slices containing 200–700-bp DNA fragments using a clean disposable blade, and then transfer the gel

slices into 0.5-ml thin-walled PCR tubes.

! CAUTION Handle the blade with appropriate protection.

39| Recover DNA fragments from the gels using an appropriate method. We have found that we achieve maximum recovery

using the ‘crush and soak’ method (Box 2).

40| Purify the eluate from Step 39 using the QIAquick PCR purification kit (or any equivalent kit) according to the

manufacturer’s protocol. Elute the DNA from the column using 50 µl of preheated EB buffer (part of the QIAquick PCR

purification kit).

41| Purify the eluate twice using 1:1-fold AMPure XP beads, as described in Box 1, and finally elute in 20 µl of

nuclease-free water.