Summary of RRBS in the lit:
- Robinson et al 2019, Atl. salmon fry
- Moghadam et al 2017, Atl. salmon fry
- Mac’s paper, WA steelhead
- Webster et al 2018, Atl. salmon fry
- Lee et al 2014, human: double digest with Taq-a1 and MspI shows 7.4% gain in CpGs in CGI regions, 41.8% gain in CpGs in non-CGI regions, 6.3% gain in CGIs and 12.7% gain in promoters
| 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
- 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
- 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
- excise fragments between 80-280bp (seems like 100-300bp would be fine)
- 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).