Ecker Lab MethyC-Seq Protocol
MethylC-seq library preparation protocol (Ecker lab)
Citation: Lister et al. Human DNA methylomes at base resolution show widespread epigenomic differences. Nature. 2009 Nov 19;462(7271):315-22.
Sonication of gDNA:
- Start with 5.3 µg gDNA
- Prepare Promega unmethylated control Lambda DNA at 25.4 ng/µl: [stock lambda DNA] = 813 ng/µl. Add 2 µl of Lambda DNA to 62 µl of Qiagen buffer AE or EB to give working conc. of 25.4 ng/µl.
- Add unmethylated lambda control DNA to 5.3 µg of sample gDNA at a final concentration of 0.5% (w/w). Make DNA solution up to 300 µl total in Qiagen buffer AE or EB. The final [gDNA] = 17.7 ng/µl for each sample.
- Sonicate (Bioruptor) gDNA for 30 sec intervals, rest for 2 min, do 5 cycles (12.5 minutes total).
- Remove tubes mix by flicking, centrifuge briefly, repeat above cycle a total of 4 times (4 x 5 cycles)
- Spin down a final time, remove 300 ng of gDNA to run on a 1% agarose gel (17 µl gDNA + 4.5µl of 5x loading dye)
- Run all samples on a gel using the 2 log DNA ladder to check sonication.
- PCR cleanup of sonicated gDNA samples:
- Follow MinElute PCR Purification Kit protocol
- Elute twice, each time in 17 µl of EB, for a total of ~32 µl elution
Perform End Repair using the End-It Kit (Epicentre):
- Add the following to the elution (Prepare a master mix of Kit contents)
o DNA sample 32 µl
o Water 2 µl
o 10x End-it Buffer 5 µl
o 10mM dNTP mix 5 µl
o 10mM ATP 5 µl
o End-it Enzyme Mix 1 µl
- Mix tube gently, spin briefly, incubate at RT for 45 min
- PCR cleanup using MinElute PCR Purification Kit, Elute in 2x 16 µl of EB.
Add “A” bases to 3’ End:
- Prepare the following reaction mix in each PCR tube
o DNA sample 32 µl
o Klenow buffer 5 µl
o 1mM dATP mix 10 µl
o Klenow exo - 3’ to 5’ exo minus 3 µl
- Mix tube gently, spin briefly, put in thermal cycler for 30 min at 37°C
- PCR cleanup using MinElute PCR Purification Kit, elute in 10 µl of EB
Prepare a 2% Agarose Gel for size selection of ligation products:
- 1.1 g of Low Range ultra agarose (Biorad)
- 55 ml of 1xTAE (no ethidium bromide)
- Boil to dissolve
- Add 2.2 µl of ethidium bromide (10 mg/ml)
Ligate Adapters to Fragments:
- Prepare the following reaction mix in each tube (from elution above)
- DNA sample 10 µl
- 2x DNA ligase buffer 25 µl
- 15 µM single end methylated adapters 10 µl
- DNA ligase 5 µl
- Mix tube gently, spin briefly, incubate for 15 min at RT
- Purify using MinElute PCR Purification Kit, elute in 2x 10 µl of EB (20 µl total)
- Add 8 µl of 4x Loading buffer to each sample.
- Prepare Low Molecular Weight ladder; 8 µl ladder + 3 µl of 4x Loading buffer
- Load gel to give max amount of space (keep libraries far apart)
- Weigh 1 empty tube for each library (for Agarose Plug), and label 175-225
- Remove tray, slide top of gel first onto blue light box.
- Use one razor blade to cut vertical (use new one for each library); cut directly
beside the lane and position the top of the razor blade at same spot on the gel
to minimize contamination between fragment sizes.
- Get new razor blade, cut at 175 bp (b/w 150 & 200 marker), cut at 225 bp (b/w
200 & 250 marker) - use same razor blade
- Use a needle to remove the slice of gel and put the 175-225bp slice in the tube.
- Repeat for each library.
- Weigh the tubes containing the 175-225 slice to calculate the weight of the
- Use the minElute Gel Extraction Kit according to the Qiagen protocol and
elute: 10 µl EB, wait 5 min, spin, 10 µl EB, wait 5 min, spin (2x 10µl EB)
- Can freeze sample overnight at -20˚C.
Bisulfite Conversion of gDNA:
- Thaw the 20 µl gDNA sample on ice
- Prepare 3 N NaOH: 1g NaOH pellets + H2O to 8.3 ml
- Prepared Solution 1+2 at 80°C (contains Sodium Bisulfite; keep away
from the light with foil).
- Perform HGS Bisulfite treatment (Human Genetic Signatures – MethylEasy
Xceed ME002) exactly as the protocol describes.
- Warm up the Solution #5 at ~70°C before the elution
- Elute in 2x 15µl Solution #5 for a total elution of 30µl (Remember to heat
sample to 95°C to finish conversion
- Whatever sample is NOT used in PCR, freeze and store at -80°C
Low Amplification (4 cycle PCR) of BS Converted gDNA:
- PCR mix:
BS Converted DNA 10 µl
10x Pfu Cx buffer 5 µl
F gDNA Primer (25uM) 1 µl
R gDNA Primer (25uM) 1 µl
dNTPs (12.5uM) 1.25 µl
H2O 30.75 µl
Pfu Turbo Cx Polymerase 1 µl
- Add 40 µl of MM to each 10µl of BS Converted DNA and amplify as follows:
1. 2 min. @ 95
2. 30 sec. @ 98
3. 15 sec. @ 98
4. 30 sec. @ 60 4 cycles (of steps 3-5)
5. 4 min. @ 72
6. 10 min. @ 72
7. Hold at 4
- Minelute PCR purify, elute samples in 2x10µl of EB, add 8µl of 4x Sample Buffer, run a 2% Agarose Gel (large gel) with a DNA ladder at 50V 1hr.
1xTAE 55 ml
Ethidium bromide 2.2 µl
- Cut out bands as before. Minelute Gel Purify the sample. Elute in 2x10µl of EB.
- Library is ready to sequence. Quantitate using Qubit dsDNA HS kit.