Summary of RRBS in the lit:

paper enzyme dig. time fragment size depth cov DMR window # DMRs
Rob MspI + Taqa1 ? 60-180 42M 6M CpGs @ 6X 500kb of diff. exp genes ~10K sites
Mog MspI + Taqa1 ? 60-180 34M didn’t say 1Mb of diff exp genes ~5.5K sites
Mac MspI o/n 100-300 38M 600K CpGs; 112K regions in all libs @ 10X 100bp region ~100 genes
Web MspI 12 hr 70-220 64M 21M CpGs; 335K @ 10X in all libs within a gene or +/- 1.5kb of TSS for promoters ~1.9K sites
Lee MspI + Taqa1 2 hr + 2 hr 80-160 5M 3.3% of CpGs @ 10X NA NA

Next steps

  1. Digest 2ug DNA
    • doing 2ug because I don’t know what the yield is going to be like after gel size-selcetion
    • get 2ug DNA in 35uL H2O
    • MSPI digest reaction mix:
      • 31.5uL MSPI (21 * 1.5uL MSP1/sample)
      • 105uL Buffer (21 * 5uL cutsmart/sample)
      • 178.5uL Water (21 * 8.5uL H2O/sample)
      • 315 = final vol.
      • add 15uL master mix/sample
    • incubate @ 37C 12 hrs
    • heat inactivate @ 80C 20 min
    • hold @ 4C
    • Taq-aI digest reaction mix:
      • 63 uL Taq-a1 (21 * 3uL Taq-a1/sample)
      • 21 uL Buffer (21*1uL cutsmart/sample)
      • 126 uL Water (21 * 6uL H2O/sample)
      • 210 = final vol.
    • add 10uL master mix/sample
    • incubate @ 65C 2 hrs
    • heat inactivate @ 80C 20 min
    • hold @ 4C
  2. Gel purify
    • excise fragments between 80-280bp (seems like 100-300bp would be fine)
      • there may be a small size range that we want to exclude, but it’s not clear what range and if its even possible. *see frag. size range info below
    • use Millipore columns
    • measure concentration with Qubit HS
    • if needed, further purify/reduce sample volume with Zymo DNA clean columns
  3. Begin zymo pico methyl kit

Notes on fragment size selection after Taq-aI/MspI digest

  • Lee paper selected 150-197bp and 207-230bp (and excluded 198-206bp) after adapter ligation
    • paper clarifies this range corresponds to 80bp-160bp fragments (before adapter ligation); suggesting adapters add 70 bp to the fragments.
    • the reason for the small size exclusion is because in silico analysis showed fragments within this size contained repetitive sequences that won’t map.
  • Mog. paper selected 150-250bp and 250-350bp after adapter ligation
    • I emailed Mog. to ask why two seperate ranges when they seem overlapping and confirm whether or not there was a fragment size exclusion
    • if the adapters used were the same length as in Lee et al, Mog. fragments pre-adapter ligation would be 80-280bp. This is pretty close to what Mac size-selected (100-300bp).