Whole-Genome Bisulfite Sequencing (WGBS) Protocol

Bisulfite Treatment

1. Prepare the Reagent by adding 900 μL water, 300 μL of M-Dilution Buffer, and 50 μL M-Dissolving Buffer. Completely dissolve the powder by mixing at room temperature for at least 10 min. Prepare prior to use.
2. Mix 130 μL of CT conversion reagent, 100 ng of DNA, and 1 ng of unmethylated lambda DNA in a PCR tube, and adjust the total volume to 150 μL with water.
3. Incubate the tubes at 98°C for 10 min and at 64°C for 150 min.
4. Load the sample to the Zymo Spin column with 600 μL of M-Binding buffer.
5. Mix the sample and M-Binding buffer by inverting the Zymo Spin column.
6. Spin at 10,000 ×g for 1 min and discard the flow through.
7. Add 200 μL M-Desulfonation buffer to the clear column, and incubate at room temperature for 15 min.
8. Transfer the column to a new tube.
9. Add 20–40 μL of M-Elution buffer directly to the column.
10. Centrifuge for 30 s at full speed to elute the DNA.
11. Use 1 μL eluent to measure the amount of DNA.

Random Priming

1. Mix the bisulfite-treated DNA, 5 μL 10× NEBuffer 2, 4 μL dNTP solution, and 1 μL 100 μM PEA2 N4 in a PCR tube, and adjust the total volume to 50 μL with water.
2. Incubate at 95°C for 3 min and at 4°C for 5 min.
3. Add 1 μL of Klenow fragment (3'–5' exo-) to the reaction.
4. Incubate the mixture at 4°C for 15 min, and increase the temperature at a rate of +1°C/min to 37°C. After maintaining the reaction at 37°C for 30 min, inactivate the enzyme by heating at 70°C for 10 min.
5. Add 50 μL AMPure XP to the reaction, keep the tube at room temperature for 5 min, place the tube on a magnetic stand to collect the beads, and remove the supernatant.
6. Add 200 μL of 300 bp cutoff solution, resuspend the beads, place the tube on a magnetic stand, and remove the supernatant.
7. Repeat the previous step once.
8. After rinsing the beads with 200 μL 70% (v/v) ethanol, elute the DNA with 12 μL of 10 mM Tris–HCl, pH8.5.
9. Measure the DNA amount.

TACS Ligation

1. Mix 10 μL of 2.5× TACS reaction buffer, 11 μL of the purified DNA in the previous step, 1 μL of 30 μM PA-anti-PEA1 P, and 1 μL of 10 mM ATP in a PCR tube.
2. Incubate at 95°C for 5 min and at 4°C for 5 min.
3. Add the 1 μL of 15 U/μL TdT and 1 μL of 100 U/μL CircLigase II to the reaction.
4. Incubate at 37°C for 30 min, 65°C for 120 min, and 95°C for 5 min.

Primer Extension (1)

1. Add 5 μL 10× Gene Taq Universal Buffer, 4 μL 2.5 mM dNTPs, 1 μL Indexing primer, 1 μL 2.5 U/μL Hot Start GeneTaq, and 14 μL water to the reaction after TACS ligation.
2. Incubate at 95°C for 3 min, 45°C for 3 min, and 72°C for 30 min.
3. Add 20 μL Buffer B2 and 5 μL 20 mg/mL proteinase K to the reaction.
4. Incubate at 50°C for 15 min.
5. Add 50 μL AMPure XP, and then incubate for 5 min at room temperature.
6. Collect the beads, and remove the supernatant.
7. Wash the beads with 200 μL cutoff solution.
8. Repeat the previous step once.
9. Rinse the beads with 200 μL 70% (v/v) ethanol.
10. Remove the residual solution completely.
11. Add 40 μL of 10 mM Tris-acetate to resuspend the beads, place the tube on the magnetic stand to separate the beads, and transfer the supernatant to a new PCR tube.
12. Measure the DNA concentration.

Primer Extension (2)

1. Add 5 μL of 10× GeneTaq Universal Buffer, 4 μL of 2.5 mM dNTPs, 1 μL of 60 μM Primer-3, and 1 μL of 5 U/μL Hot Start GeneTaq to 39 μL of the purified DNA in the previous step.
2. Incubate at 94°C fo r3 min, 45°C for 5 min, and 72°C for 30 min.
3. Add 50 μL AMPure XP and incubate at room temperature for 5 min.
4. Collect the beads by placing the tube on a magnetic stand.
5. Wash the beads with 200 μL cutoff solution.
6. Repeat the step once.
7. Rinse the beads with 200 μL 70% (v/v) ethanol.
8. Remove the residual solution completely.
9. Add 26 μL of 10 mM Tris-acetate to resuspend the beads, place the tube on the magnetic stand to separate the beads, and transfer the supernatant to a new PCR tube.
10. Take 1 μL of the purified DNA to measure the concentration.

Library QC

1. Thaw the contents of the Library Quantitation kit at room temperature.
2. Prepare a master mix solution by mixing 10 μL/well of Terra PCR Direct TB Green Premix (2×), 4 μL/well of 5× Primer Mix, 0.4 μL/well of 50× ROX Reference Dye, and 3.6 μL/well of water to prepare a master mix solution. Multiply the volume of each reagent with the number of wells.
4. Dispense 18 μL of the master mix into every PCR tube.
5. Dilute the libraries with 10 mM Tris–HCl (pH 0.0) at appropriate dilution rates.
6. Add either 2 μL of templates, i.e., standard, non-templated control, or diluted libraries, to a PCR tube containing the master mix.
7. Prepare real-time PCR machine.
8. Perform PCR amplification with the following program: 95°C for 1 min; 35 cycles of three-step incubations at 95°C for 10 s, 60°C for 15 s, and 68°C for 45 s.
9. Prepare an electrophoresis device.
10. Mix 1 μL of PCR-amplified DNA with 10 μL of denaturing loading dye, incubate at 70°C for 5 min, and load 5 μL of sample on a 6% Novex TBE-Urea gel.
11. Run electrophoresis at 300 V.
12. Stain the gel with SYBR Gold nucleic acid gel stain, and take photograph.

Sequencing

1. Mix the libraries appropriately.
2. Adjust the concentration.
3. Run sequencer.

Reference:

1. Miura F, Ito T. Post-bisulfite Adaptor Tagging with a Highly Efficient Single-Stranded DNA Ligation Technique[M]//Transcription Factor Regulatory Networks. Humana, New York, NY, 2023: 45-57.