Post-bisulfite Adaptor Tagging Protocol Revision 12
November 7, 2013 (Thr.)

ReagentsQubit dsDNA BR Assay Kit (LifeTechnologies, # Q32850)

Qubit dsDNA HS Assay Kit (LifeTechnologies, # Q32851)



Qubit ssDNA Assay Kit (LifeTechnologies, # Q10212)

Agencourt AMPure XP (Beckman Coulter, # A63880)

Conventional 10× PCR Buffer (100 mM Tris-HCl [pH 8.3], 500 mM KCl, 15 mM MgCl2)



Agilent RNA 6000 Pico Kit (Agilent, #5067-1513)



10 mM Tris-acetate, pH 8.0



10 mM Tris-HCl, pH 7.5



Klenow Fragment (3′→5′ exo−) (NEB, #M0212M)
Be sure to use the enzyme provided at 10-fold higher concentration (i.e., #M0212M; 50,000 U/mL)
than the conventional ones (i.e., #M0212S and #M0212L; 5,000 U/mL)

Bst DNA Polymerase Large Fragment (NEB, #M0275S)



Exonuclease I (NEB, #M0293S）



Phusion Hot Start High-Fidelity DNA Polymerase (Finnzyme, #F-540S)

EZ DNA Methylation-Gold Kit (ZYMO Research, #D5005)

Dynabeads M-280 Streptavidin (LifeTechnologies, #112-05D)

2× BW(Li) Solution (50 mL)


LiCl*

6.3 g



1 M Tris-HCl, pH 8.0

0.5 mL

500 mM EDTA

0.1 mL*The process of dissolution of LiCl is exothermic. To avoid bumping of the solution, dissolve LiCl in
40 mL of double-distilled water (ddH2O) at a time. After complete dissolution of LiCl, add Tris and
EDTA, and adjust the volume to 50 mL with ddH2O.



0.1 N NaOH Solution
Dilute 10 N NaOH stock solution before use.



KAPA Library Quantification Kit for Illumina (KAPA, # KK4824 or qPCR platform
specific ones)Hybridization Buffer A (50 mL)

5 M NaCl

9 mL(final 900 mM)

1 M Tris-HCl, pH 7.4

9 mL(final 180 mM)



ddH2O

32 mL
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Post-bisulfite Adaptor Tagging Protocol Revision 12
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Oligonucleotides (OPC grade)

Bio-PEA2-N4 (100 µM)
5′-biotin-ACA CTC TTT CCC TAC ACG ACG CTC TTC CGA TCT NNN N-3′



PE-reverse-N4 (100 µM)
5′-CAA GCA GAA GAC GGC ATA CGA GAT NNN N-3′



PBAT-PE-iX-N4 (100 µM each)5′-CAA GCA GAA GAC GGC ATA CGA GAT XXX XXX GTA AAA CGA CGG
CCA GCA GGA AAC AGC TAT GAC NNN N-3′
Replace the underlined hexamer with each of the following index sequences, which arecomplementary to those used in the adaptors used in TruSeq DNA LT Sample Prep Kit, so that thesame index numbers as those in the kit can be used. The index numbers 17, 24 and 26 are reserved
by Illumina for unknown reasons.

Index #

Sequence

Index #

Sequence

Index #

Sequence

1

CGTGAT

9

CTGATC18

GCGGAC

2

ACATCG10

AAGCTA

19

TTTCAC3GCCTAA

11

GTAGCC20GGCCAC

4TGGTCA

12

TACAAG21

CGAAAC

5

CACTGT

13

TTGACT

22

CGTACG

6

ATTGGC14

GGAACT

23

CCACTC7GATCTG15TGACAT

25

ATCAGT

8

TCAAGT

16

GGACGG27

AGGAAT

Primer 3 (100 µM)
5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CACGAC GCT CTT CCG ATC T-3′
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Post-bisulfite Adaptor Tagging Protocol Revision 12
November 7, 2013 (Thr.)

PBAT-PE-Seq (100 µM)
5′-GTA AAA CGA CGG CCA GCA GGA AAC AGC TAT GAC-3′



PBAT-PE-Idx (100 µM)
5′-GTC ATA GCT GTT TCC TGC TGG CCG TCG TTT TAC-3′

Plastic disposables


Tubes




Use low-retention 1.5- and 0.2-mL tubes in all the steps.

Tips
Use low-retention tips for dispensing streptavidin-coated magnetic beads.

Equipment


DynaMag-2 Magnet (LifeTechnologies) or its equivalent

SPRIPlate 96R Magnet Plate (Beckman Coulter) or its equivalent

TOMY MX-301 High-Speed Refrigerated Microcentrifuge or its equivalent

Agilent Bioanalyzer 2100

Qubit Fluorometer or Qubit 2.0 Fluorometer (Life Technologies)

TOMY PMC-060 Capsulefuges or its equivalent

ABI9700 thermal cycler or its equivalent

StepOnePlus Real-Time PCR System (Applied Biosystems) or its equivalent

XCell SureLock Mini-Cell (Life Technologies, # EI0001) or its equivalentPower supply for electrophoresis

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Post-bisulfite Adaptor Tagging Protocol Revision 12
November 7, 2013 (Thr.)

Programs for thermal cycling








Program 1 (for bisulfite treatment, see Section II)
1.

98°C for 10 min

2.

64°C for 150 min

3.

Soaking at 4°C

Program 2 (for first-strand synthesis, see Section III)1.

94°C for 5 min

2.

4°C for 20 min

3.

Gradual increment from 4°C to 37°C at a rate of +1°C/min*

4.

37°C for 90 min

5.

70°C for 10 min

6.

Soaking at 4°C

Program 3 (for second-strand synthesis, see Section VI)
1.

94°C for 5 min

2.

4°C for 20 min

3.

Gradual increment from 4°C to 37°C at a rate of +1°C/min*

4.

37°C for 30 min

5.

70°C for 10 min

6.

Soaking at 4°C

Program 4 (for elution, see Section VII)
1.

94°C for 5 min

2.

55°C for 15 min

3.

68°C for 30 min

4.

Soaking at 4°C*If your thermal cycler cannot generate a temperature ramp of +1°C/min, you may use a two-step PCR
cycling program with an increment of temperature by 1°C for every step. If the first cycle of the programis set as 4.0°C for 30 sec followed by 4.5°C for 30 sec, then the temperature will reach to 37°C after 33
cycles with the intended rate of +1°C/min.

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Post-bisulfite Adaptor Tagging Protocol Revision 12
November 7, 2013 (Thr.)I. Estimation of DNA concentration (Day 1)
Accurate estimation of DNA concentration is critical. We routinely use Qubit dsDNA BR Assay Kit for the
purpose. Do not measure OD260, because various materials other than DNA absorb light at 260 nm,
leading to an overestimation of DNA concentration.
1.

Measure DNA concentration of sample DNA with Qubit dsDNA BR Assay Kit and Qubit
Fluorometer according to the manufacturer’s instruction.

II. Bisulfite treatment (Day 1)
We routinely start with 100 ng of DNA, because this amount is easy to handle. However, note that the
maximum efficiency of template preparation is achieved with ~1 ng of DNA as a starting material. Thus,
the bisulfite-treated DNA may be divided into several aliquots before first-strand synthesis to further
increase the efficiency of template preparation.

1.

Add 900 µL of ddH2O, 50 µL of M-Dissolving Buffer, and 300 µL of M-Dilution Buffer to
one tube of CT Conversion Reagent.
Use freshly prepared CT conversion reagent to ensure high yield and efficient bisulfite conversion.

2.

Dissolve the material by rotating the tube of CT Conversion Reagent for 10 min at room
temperature.

3.

Mix the following components well.
CT Conversion Reagent

130 µL(20 − x) µL

ddH2O
Sample DNA

x µL

4.

Divide the solution into three 50-µL aliquots in 0.2-mL tubes.

5.

Place the tubes on a thermal cycler, and start Program 1.

6.

Place a Zymo-Spin™ IC Column in a Collection Tube and add 600 µL of M-Binding
Buffer to the column.

7.

Add the sample from Step 5 to the M-Binding Buffer in the column. Close the cap and mix
by inverting several times.

8.

Centrifuge at full speed (≥10,000 × g) for 30 sec.

9.

Reload the flow-through onto the same column again.

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Post-bisulfite Adaptor Tagging Protocol Revision 12
November 7, 2013 (Thr.)

We occasionally encountered "shunts" in the column, through which the solution flows with minimal
contact with the resin. Thus, we recommend to carefully inspect the column before use. Reloading of
the flow-through would help the solution to have enough contact with the resin.

10. Centrifuge at full speed (≥10,000 × g) for 30 sec.
11. Discard the flow-through.
12. Add 100 µL of M-Wash Buffer prepared with ethanol to the column, and centrifuge at fullspeed for 30 sec. Discard the flow-through.
13. Add 200 µL of M-Desulphonation Buffer to the column, and let the column stand at room
temperature for 15 min.
14. Centrifuge at full speed for 30 sec. Discard the flow-through.
15. Add 200 µL of M-Wash Buffer with ethanol to the column, and centrifuge at full speed for
30 sec. Discard the flow-through.
16. Repeat the wash in Step 15 once more, and transfer the spin column to a new 1.5-mL tube.
17. Add 22 µL of M-Elution Buffer directly to the column matrix and let the column stand atroom temperature for 2 min. Centrifuge at full speed for 30 sec to elute the DNA.
The elusion volume (22 µL) includes 1 µL for determination of yield by Qubit ssDNA Assay Kit and 1 µL
for quality check (QC) with Agilent Bioanalyzer with RNA 6000 Pico Kit. Typically, the yield of DNA isbetween 30% and 70% of the input, and the size range of denatured DNA is 100~1,000 nt with a peakaround 600 nt. When the starting amount of DNA is <30 ng, both platforms will fail to detect the eluted
DNA. Thus, omit these QC steps and reduce the elution volume in Step 16 to 20 µL.
Never stop here. Proceed immediately to the first-strand synthesis step, because the bisulfite-treated DNA
is labile.

III. First-strand synthesis (Day 1)
1.

2.

Prepare the first-strand synthesis reaction mix as follows.
10× NEBuffer 25 µL

2.5 mM dNTPs

5 µL

ddH2O

16 µL

Bio-PEA2-N4 (100 µM)

4 µL

Bisulfite-treated sample DNA

20 µL

Place the tube on a thermal cycler and start Program 2.
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Post-bisulfite Adaptor Tagging Protocol Revision 12
November 7, 2013 (Thr.)

3.

After 5 min from starting step 2 of Program 2 (i.e., incubation step at 4°C), pause theprogram and remove the tube from the thermal cycler. Add 1.5 µL of Klenow Fragment(3′→5′ exo−) to the first-strand synthesis solution and mix well.

4.

Place the tube back on the thermal cycler and resume the Program 2 to complete thefirst-strand synthesis reaction.

You can stop here by either leaving the tube at 4°C or storing it at −20°C until use. This is presumably
because the bisulfite-treated DNAs are now double-stranded and excessive primers in the solution serve
as a carrier DNA to prevent the adsorption of template DNA to the tube wall.

IV. Removal of excess primers (Day 2)
1.

Transfer the solution of the first strand reaction (~50 µL) into a new 1.5-mL tube, add 50µL of AMPure XP, mix well, and spin the tube briefly.
At this mixing ratio (i.e., DNA solution:AMPure XP = 1:1), DNA fragments shorter than 200 bp are
effectively removed in the supernatant. While primers and primer dimers are shorter than 100 nt, theproducts of the first-strand synthesis are longer than 200 bp.

2.

Let the tube stand at room temperature for 10 min.

3.

Place the tube on a magnetic stand and wait for the beads to be collected. Then, remove thesupernatant carefully so as not to aspirate the beads.

4.

Add 200 µL of 75% ethanol to wash the beads and then remove the supernatant.

5.

Add 45 µL of 10 mM Tris-acetate (pH 8.0) and vortex the tube well to disperse the beads.
Following a brief centrifugation, place the tube on the magnetic stand and wait for thebeads to be collected.

6.

Transfer the supernatant into a new 1.5-mL tube. Add 5 µL of 10× PCR Buffer and 50 µL
of AMPure XP to the supernatant. Then mix well and spin briefly.
Addition of 10× PCR Buffer at this step increases the reproducibility and yield of AMPure XP-based
purification of DNA.

7.

Let the tube stand at room temperature for 10 min.

8.

Place the tube on the magnetic stand and wait for the beads to be collected. Then, remove
the supernatant carefully so as not to aspirate the beads.
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Post-bisulfite Adaptor Tagging Protocol Revision 12
November 7, 2013 (Thr.)9.

Add 200 µL of 75% ethanol to wash the beads and then remove the supernatant.

10. Add 51 µL of 10 mM Tris-acetate (pH 8.0) and vortex the tube well to disperse the beads.
After a brief centrifugation, place the tube on the magnetic stand and wait for the beads tobe collected.
11. Save the supernatant in a new 1.5-mL tube.
12. Use 1 µL of the supernatant to measure DNA concentration by the Qubit dsDNA HS Kit.
Typical yield at this step is between 40% and 60% of the input. When starting from <30 ng of DNA, thekit will fail to quantify the DNA. Thus, omit this QC step and reduce the elution volume in Step 10 to 50µL.

V. Capturing biotinylated DNA on streptavidin beads (Day 2)
1.

Take 20 µL of well-dispersed suspension of Dynabeads M280 Streptavidin beads into a
1.5-mL tube. Place the tube on the magnet stand to collect the beads.

2.

Remove the supernatant, and add 50 µL of 2x BW(Li) buffer to suspend the beads.

3.

Add the suspension of beads to the product obtained in Section IV.

4.

Incubate the tube at room temperature for 30 min with gently rotating the tube.

5.

Place the tube on the magnet stand to collect the beads, and then remove the supernatant.

6.

Add 180 µL of 2× BW(Li) buffer to the beads, vortex well, and spin the tube briefly.

7.

Place the tube on the magnet stand to collect the beads, and then remove the supernatant.

8.

Suspend the beads in 180 µL of 0.1 N NaOH solution, vortex well, incubate at room
temperature for 2 min, and spin briefly.

9.

Place the tube on the magnet stand to collect the beads, and then remove the supernatant.

10. Repeat steps 8 and 9 once again.
11. Add 180 µL of 2× BW(Li) buffer to the beads, vortex well, and spin the tube briefly.
12. Place the tube on the magnet stand to collect the beads, and then remove the supernatant.
13. Add 180 µL of 10 mM Tris-HCl (pH 7.5) to the beads, vortex well, and spin the tube
briefly.

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Post-bisulfite Adaptor Tagging Protocol Revision 12
November 7, 2013 (Thr.)

VI. Second-strand synthesis (Day 2)
1.

Place the tube on the magnet stand to collect the beads and remove the supernatant.

2.

Prepare the second-strand synthesis reaction mix as follows and add to the beads.
10× NEBuffer 25 µL

2.5 mM dNTPs

5 µL

ddH2O

36 µL

PE-reverse-N4 (100 µM) for single-end sequencing, or

4 µL

PBAT-PE-iX-N4 (100 µM) for paired-end and index sequencing

3.

Suspend the beads by vortexing and transfer the beads suspension into a new 0.2-mL tube.

4.

Place the tube on the thermal cycler and start Program 3.

5.

After 5 min of starting step 2 of Program 3 (i.e. incubation step at 4°C), pause the program
and remove the tube from the thermal cycler. Add 1.5 µL of Klenow Fragment (3′→5′exo−) to the second-strand synthesis solution and mix well.

6.

Place the tube back on the thermal cycler and resume the Program 3 to complete thesecond-strand synthesis reaction.

VII. Chase reaction (Day 2)
1.

Place the tube on a magnet stand to collect the beads and remove the supernatant.

2.

Prepare the chase reaction mix as follows and added to the beads.

3.

10× ThermoPol Buffer

5 µL

2.5 mM dNTPs

5 µL

ddH2O

40 µL

Bst DNA Polymerase Large Fragment

1 µL

Incubate the reaction at 65°C for 30 min.

VIII. Elution/extension of template DNA (Day 2)
1.

Place the tube on the magnet stand to collect the beads and remove the supernatant.

2.

Prepare the elution/extension reaction as follows and added to the beads.
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Post-bisulfite Adaptor Tagging Protocol Revision 12
November 7, 2013 (Thr.)

5× Phusion HS Buffer

10.0 µL

2.5 mM dNTPs

5 µL

ddH2O

35 µL

Primer 3 (100 µM)

0.4 µL

Phusion Hot Start High-Fidelity DNA Polymerase

1 µL

This step not only enables the precise selection of double-stranded DNA by SPRI beads, butalso synthesizes the sequence required for bridge PCR.
3.

Start Program 4

4.

Place the tube on the magnet stand to collect the beads, and transfer the supernatant into a
new 1.5-mL tube.

5.

Add 1 µL of Exonuclease I to the supernatant, mix well, and incubate the tube at 37°C for
30 min followed by heat-inactivation at 70°C for 10 min.

6.

Use 1 µL of eluted DNA and Qubit dsDNA HS Kit to measure the concentration of DNA.

IX. Size fractionation (Day 2)
1.

Add 50 µL of AMPure XP to the eluted DNA (50 µL), mix well, and spin briefly.

2.

Place the tube on the magnetic stand and wait for the beads to be separated. Then, remove
the supernatant carefully so as not to aspirate the beads.

3.

Add 200 μL of 75% ethanol to wash the beads and then remove the supernatant.

4.

Add 45 μL of 10 mM Tris-acetate (pH 8.0) and vortex the tube well to disperse the beads.
After a brief centrifugation, place the tube on the magnetic stand and wait for the beads tobe collected.

5.

Transfer the supernatant into a new 1.5-mL tube. Add 5 µL of 10× PCR Buffer and 50 µL
of AMPure XP to the supernatant. Mix well and spin briefly.

6.

Let the tube stand at room temperature for 10 min.

7.

Place the tube on the magnetic stand and wait for the beads to be collected. Then, remove
the supernatant carefully so as not to aspirate the beads.

8.

Add 200 µL of 75% ethanol to wash the beads and remove the supernatant.

10

Post-bisulfite Adaptor Tagging Protocol Revision 12
November 7, 2013 (Thr.)9.

Add 22 µL of 10 mM Tris-acetate (pH 8.0), and vortex the tube well to disperse the beads.
Following a brief centrifugation, place the tube on the magnetic stand and wait for thebeads to be collected.

10. Transfer the supernatant to a new 1.5-mL tube.
11. Use 1 µL of the supernatant to measure the concentration of DNA using the Qubit dsDNA
HS Kit.
Typical yield at this step is between 20% and 40% of the input DNA. When the starting amount of DNA is<30 ng, the kit will fail to quantify DNA. Thus, omit this QC step and reduce the elution volume in Step 9
to 21 µL.

X. qPCR quantitation of template DNA (Day 3)
1.

Determine the exact molar concentration of template DNA using an appropriate qPCR
assay.

Note that the product obtained in Step IX contains not only intact sequencing templates but also
several-fold greater amounts of byproducts.
It is thus essential to determine the correct concentration of the template DNA by qPCR but not byfluorometry. We use Library Quantification Kits for Illumina (KAPA biosystems) according to the
manufacturer’s instruction for the quantification, because this kit is easy to use and highly reproducible.
Typical mass yield at this step is calculated to be 2~8% relative to the starting amount of DNA. If thestarting DNA is 100 ng, the amount of obtained template is ~20 fmol, which is sufficient for sequencing in
20 lanes of Illumina HiSeq 2000 or 4 runs of MiSeq.
In addition, the byproducts also make it impossible to directly examine the size of template DNA byelectrophoresis. Accordingly, we examine the size of qPCR product. Note that, once PCR reaches to theplateau, the size of PCR-amplified templates cannot be accurately examined under non-denaturingconditions. Thus, we use the standard polyacrylamide/urea/TBE gel electrophoresis. The size is typically200~500 nt with a peak around 300 nt.

11Post-bisulfite Adaptor Tagging Protocol Revision 12
November 7, 2013 (Thr.)

XI. Sequencing (Day 3)
For paired-end and index sequencing, be sure to add PBAT-PE-Seq and PBAT-PE-Idx to the Illumina
primer mixes for read 2 and index, respectively, at the final concentration of 0.5 μM.

Calculation of template volume required for sequencing
1.

Calculate the volume of template required in cluster generation with the following equationand parameters.
HiSeq 2000 or

HiSeq 2500

HiSeq 2500

HiSeq 2500

rapid run mode

rapid run modewith cBot

without cBot or

Platformhigh output

MiSeq

mode

Target concentration of denatured

10*

template (pM)
Target volume of template (µL)

120

Molar concentration of template

70

480

y

(pM) determined in Section X
120 × 10𝑦Volume of template required= x (µL)

70 × 10𝑦

480 × 10𝑦*For the target concentration, 10 pM is a good point to start optimization.

For a single lane of Illumina HiSeq 2000 or HiSeq 2500 in high output
mode using cBot
1.

Dispense 100 µL, plus extra volume for pipetting loss, of Hybridization Buffer A to a new
1.5-mL tube and place it on ice.

2.

Prepare 2 N NaOH by diluting 10 N NaOH solution.

3.

Denature sequencing templates as follows.
Template DNA solution

x µL(19 − x) µL

ddH2O
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Post-bisulfite Adaptor Tagging Protocol Revision 12
November 7, 2013 (Thr.)

2N NaOH

1 µL

4.

Let the tube stand at room temperature for 5 min.

5.

Add 100 µL of Hybridization Buffer A to the denatured template, mix well, and place thetube on ice.

6.

Start cluster generation according to the manufacturer’s instruction.

Since bisulfite-converted DNA has an extremely-biased base composition, each flow cell must
include a control lane for the phiX control template to enable correct color normalization offluorescent signals. Alternatively, the phiX control can be added to the sample as described
below.

For a single lane of Illumina HiSeq 2500 in rapid run mode using cBot
1.

Dispense 58 µL, plus extra volume for pipetting loss, of Hybridization Buffer A to a new
1.5-mL tube and place it on ice.

2.

Prepare 2 N NaOH by diluting 10 N NaOH solution.

3.

Denature sequencing templates as follows.
Template DNA solution

x µL(11 − x) µL

ddH2O
2N NaOH

0.6 µL4.

Let the tube stand at room temperature for 5 min.

5.

Add 58 µL of the ice-cooled Hybridization Buffer A to the denatured template, mix well,and place the tube on ice.

6.

Add 70 µL of ice-cold 8 pM denatured phiX control to the tube and mix well.
For the same reason as described above, the phiX control template must be added to thesample. Follow the instruction provided by Illumina for the preparation of denatured phiX
control template.

7.

Start cluster generation according to the manufacturer’s instruction.
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Post-bisulfite Adaptor Tagging Protocol Revision 12
November 7, 2013 (Thr.)

For Illumina HiSeq 2500 in rapid run mode without cBot or Illumina
MiSeq
1.

Dispense 400 µL, plus extra volume for pipetting loss, of Hybridization Buffer A to a new
1.5-mL tube and put it on ice.

2.

Prepare 2 N NaOH by diluting 10 N NaOH solution.

3.

Denature sequencing templates as follows.
Template DNA solution

x µL(76 − x) µL

ddH2O
2N NaOH

4 µL

4.

Let the tube stand at room temperature for 5 min.

5.

Add 400 µL of ice-cold Hybridization Buffer A, mix well and place the tube on ice.

6.

Add 120 µL of ice-cold 8 pM denatured phiX control to the tube and mix well.
For the same reason as described above, the phiX control template must be added to thesample. Follow the instruction provided by Illumina for the preparation of denatured phiX
control template.

7.

Start run according to the manufacturer’s instruction.

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Post-bisulfite Adaptor Tagging Protocol Revision 12
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References
Miura, F., Enomoto, Y., Dairiki, R. & Ito, T. (2012) Amplification-free whole genome bisulfite
sequencing by post-bisulfite adaptor tagging. Nucleic Acids Res. 40, e136.
Kobayashi, H., Sakurai, T., Miura, F., Imai, M., Mochizuki, K., Yanagisawa, E., Sakashita, A.,
Wakai, T., Suzuki, Y., Ito, T., Matsui, Y. & Kono, T. (2013) High resolution DNA methylome
analysis of primordial germ cells identifies gender-specific reprogramming in mice. Genome
Res. 23, 616–627.
Shirane, K., Toh, H., Kobayashi, H., Miura, F., Chiba, H., Ito, T., Kono, T. & Sasaki, H. (2013)
Mouse oocyte methylomes at base resolution reveal genome-wide accumulation of non-CpG
methylation and role of DNA methyltransferases. PLoS Genet. 9, e1003439.

15