Bisulfite Sequencing of 5-methylcytosine
DNA Fragmentation
See genomic DNA sequencing protocol.
Blunt-end and size selection
- 1. Assemble the following reaction mix
| Fragmented genomic DNA | 10 ul |
| 10 x T4 Ligase Buffer | 5 ul |
| Polynucleotide kinase | 1 ul |
| Mung Bean Nuclease | 1 ul |
- 2. Incubate at 20 C for 30 min.
- 3. Size-fraction on agarose gel, cut the 100-200 bp band.
dA-tailing
- 4. Prepare the reaction as follow:
| End-repaired DNA | 20 ul |
| 10 x Ligase Buffer | 2.5 ul |
| Klenow 3’-5’ exo- | 2 ul |
| T4 PNK | 0.5 ul |
- 5. Incubate at 37C for 30 min.
- 6. Purify DNA using AMPure XP.
5mC PE adapter Ligation
- 7. Assemble ligation reaction on ice as follow:
| DNA | 22 ul |
| 5mC PE adapters (50 uM) | 1 ul |
| 2x Ligation Buffer | 25 ul |
| T4 DNA Ligase (Enzymatics) | 2 ul |
- 5. Incubate at 20C for 15 min.
- 6. Purify DNA using AMPure XP.
2x Bisulfite treatment (5 h)
- 10. Assemble the BS reaction (follow Qiagen protocol).
- 11. Purify DNA Using the yellow qiagen column.
- 12. Elute 2 x 20 ul water.
- 13. Use 1 ul for Q-PCR or conventional PCR for 20 cycle to check the library quality.
Pfu Turbo Cx PCR
- 14. Assemble PCR reaction on ice as follow:
| S-treated DNA | 10 ul |
| 10x Pfu Turbo Cx buffer | 5 ul |
| 50 mM MgCl2 | 0.5 ul |
| dNTP | 1 ul |
| PE primer mix (10 uM) | 2 ul |
| Pfu Turbo Cx | 1 ul |
| Water | to 50 ul |
- 15. Purify library using AMPure XP beads.
- 16. QC by Agilent Bioanalyzer.
Methylated adapter oligos:
- Adapter oligo A: phos-GAT[5Me-dC]GGAAGAG[5Me-dC]GGTT[5Me-dC]AG[5Me-dC]AGGAATG[5Me-dC][5Me-dC]GAG
- Adapter oligo B: A[5Me-dC]A[5Me-dC]T[5Me-dC]TTT[5Me-dC][5Me-dC][5Me-dC]TA[5Me-dC]A[5Me-dC]GA[5Me-dC]G[5Me-dC]T[5Me-dC]TT[5Me-dC][5Me-dC]GAT[5Me-dC]T
|