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

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mixture at 9,000g for 1 min at 4 °C.

component amount per reaction (ml) Final amount
10 U/µl MspI 0.9 9 U
10× Tango buffer 1.8 1×
Unmethylated λ-DNA (60 fg/µl) 1 60 fg
Nuclease-free water up to 13 —
 cr ItIcal step Always ensure that the λ spike-in accounts for ~1% (mass/mass) of the total genomic DNA of every picked
single cell. For example, if you are starting with a single diploid mammalian cell, add 1 µl (~60 fg) of unmethylated λ-DNA to
the digestion mixture.


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

## Page 7

protocol


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 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 (ml) Final amount
30 Weiss U/µl T4 DNA ligase 1 30 Weiss U
10× Tango buffer 0.5 1×
100 mM ATP 0.25 1 mM
1:20 diluted adapter 1 —
Nuclease-free water up to 5 —
 cr ItIcal 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)

mixture at 9,000g for 1 min at 4 °C.

component amount per reaction (ml) Final amount

10 U/µl MspI 0.9 9 U

10× Tango buffer 1.8 1×

Unmethylated λ-DNA (60 fg/µl) 1 60 fg

Nuclease-free water up to 13 —

 cr ItIcal step Always ensure that the λ spike-in accounts for ~1% (mass/mass) of the total genomic DNA of every picked

single cell. For example, if you are starting with a single diploid mammalian cell, add 1 µl (~60 fg) of unmethylated λ-DNA to

the digestion mixture.

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

## Page 7

protocol

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 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 (ml) Final amount

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

10× Tango buffer 0.5 1×

100 mM ATP 0.25 1 mM

1:20 diluted adapter 1 —

Nuclease-free water up to 5 —

 cr ItIcal 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)