11.4.5. Bisulfite conversion and amplification of the final library

  1. Bisulfite treatment can be carried out with the EZ-DNA Methylation Kit (Zymo) as recommended by the manufacturer, with the exception of a modified thermal profile for the bisulfite conversion reaction. Alternatively the QIAGEN EpiTect Bisulfite Kit can be used.
    The conversion is carried out in a thermal cycler using the following thermal profile:
    15 cycles of:
    95° C for 15 sec,
    50° C for 1hr,
    Incubate at 4° C for at least 10 min.
  2. Amplify the resulting libraries using the Fast Start High Fidelity PCR System (Roche, 03 553 400 001) with buffer 2, the Illumina PE1.1 and PE2.1 amplification primers, and the below protocol.
    PCR thermal profile:
    95°C- 2min,
    11 cycles of:
    95°C for 30 sec,
    65°C for 20 sec,
    72°C for 30 sec,
    72°C for 7 min,
    20°C hold.
  3. Purify products on PCR purification columns (MinElute, Qiagen), eluting in 20µl elution buffer (Qiagen).