Ecker Lab MethyC-Seq Protocol

Procedure : 

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

   agarose plug

-  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:

 

Components:                                                1x                       

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

                                                Total:            50µl

 

- Add 40 µl of MM to each 10µl of BS Converted DNA and amplify as follows:

 

PCR Setup

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.

 

2% Gel:

Agarose                        1.1g

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.

 

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