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

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11\| Add 13 µl of MspI digestion mixture from Step 10 to each tube from Step 9 so that the total volume of each sample is
18 µl: 4.8 µl of lysis buffer, 0.2 µl of carry-over PBS-BSA (single cell included) and 13 µl of MspI digestion mixture. Mix well
by vortexing the tubes, and centrifuge them at 9,000g for 1 min at 4 °C.

12\| Incubate the reaction at 37 °C for 3 h, and then at 80 °C for 20 min to inactivate the restriction enzyme. Centrifuge the
tubes at 9,000g for 1 min at 4 °C, and place the tubes on ice immediately. Proceed to the next step immediately.

End-repair/dA-tailing reaction ● TIMING 1–2 h
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 (ml) Final amount
5 U/µl Klenow Fragment, exo– 1 5 U

10× Tango buffer 0.2 1×
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 thereserved. tubes, and centrifuge the mixture at 9,000g for 1 min at 4 °C.
rights 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
All at 9,000g for 1 min at 4 °C, and place the tubes on ice immediately.
Inc. 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.America,
Component Amount per reaction (ml) Final amount
30 Weiss U/µl T4 DNA ligase 1 30 Weiss UNature
2015 10× Tango buffer 0.5 1×
© 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 well by vortexing, and then centrifuge the mixture at 9,000g for 1 min at 4 °C.

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.

nature protocols \| VOL.10 NO.5 \| 2015 \| 651

## Page 8

protocol

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 (ml) 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 nMreserved.
rights 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
All stalling, thus ensuring that both methylated and unmethylated DNA fragments will be amplified with similar efficiencies.
? TROUBLESHOOTINGInc.

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 mixAmerica, 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.
Nature 26\| Perform 25 cycles of PCR on the PCR thermocycler and run the following program:
2015 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

11| Add 13 µl of MspI digestion mixture from Step 10 to each tube from Step 9 so that the total volume of each sample is

18 µl: 4.8 µl of lysis buffer, 0.2 µl of carry-over PBS-BSA (single cell included) and 13 µl of MspI digestion mixture. Mix well

by vortexing the tubes, and centrifuge them at 9,000g for 1 min at 4 °C.

12| Incubate the reaction at 37 °C for 3 h, and then at 80 °C for 20 min to inactivate the restriction enzyme. Centrifuge the

tubes at 9,000g for 1 min at 4 °C, and place the tubes on ice immediately. Proceed to the next step immediately.

End-repair/dA-tailing reaction ● TIMING 1–2 h

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 (ml) Final amount

5 U/µl Klenow Fragment, exo– 1 5 U

10× Tango buffer 0.2 1×

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 thereserved. tubes, and centrifuge the mixture at 9,000g for 1 min at 4 °C.

rights 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

All at 9,000g for 1 min at 4 °C, and place the tubes on ice immediately.

Inc. 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.America,

Component Amount per reaction (ml) Final amount

30 Weiss U/µl T4 DNA ligase 1 30 Weiss UNature

2015 10× Tango buffer 0.5 1×

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 well by vortexing, and then centrifuge the mixture at 9,000g for 1 min at 4 °C.

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.

nature protocols | VOL.10 NO.5 | 2015 | 651

## Page 8

protocol

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 (ml) 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 nMreserved.

rights 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

All stalling, thus ensuring that both methylated and unmethylated DNA fragments will be amplified with similar efficiencies.

? TROUBLESHOOTINGInc.

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 mixAmerica, 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.

Nature 26| Perform 25 cycles of PCR on the PCR thermocycler and run the following program:

2015 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