Finished EtOH precipitation of MeDIP gDNA. Samples were pelleted 16,000g, 4C, 30mins. Supe was discarded. Washed with 1mL 70% EtOH, pelleted 16,000g, 4C, 15mins. Supe discarded. MeDIP DNA was resuspended in 100uL of TE (pH = 8.5). Wash samples, containing unmethylated DNA, were resuspended/combined in a total of 100uL TE (pH = 8.5). Samples were spec’d:
R37: MeDIP DNA = 1.393ug recovery. This is ~13% of the input total gDNA (11.25ug) and is ~28% of the total DNA recovered in the procedure (4.935ug). Unmethylated DNA = 3.542ug total recovery. This is ~31% of the input total gDNA (11.25ug) and is ~72% of the total DNA recovered in the procedure (4.935ug). Total DNA recovery = ~44%.
R51: MeDIP DNA = 1.256ug recovery. This is ~14% of the input total gDNA (8.75ug) and is ~23% of the total DNA recovered in the procedure (5.462ug). Unmethylated DNA = 4.206ug total recovery. This is ~48% of the input total gDNA (8.75ug) and is ~77% of the total DNA recovered in the procedure (5.462ug). Total DNA recovery = ~62%.
There definitely seemed to be a high degree of salt carryover from the procedure, despite the phenol:chloroform treatment and EtOH precipitation. As such, I believe this is the reason that the 260/230 ratios are so out of whack. Possibly explains why the 260/280 ratios for the MeDIP DNA are so high, too?
These results demonstrate what we can expect to recover from this procedure, as well as how much DNA gets lost during processing. MeDIP DNA and unmethylated DNA were stored @ -20C.
Continued MeDIP process from yesterday. Protein A/G beads were pelleted XXXXXXXXX, supe transferred to clean tube. Beads were washed 3x in the following fashion, each wash saved to retain unmethylated DNA:
Samples were phenol:chloroform extracted and EtOH precipitated:
Continued MeDIP process from yesterday. Added 20uL of Protein A/G Plus Agarose (Santa Cruz Biotech) beads to each sample and continued incubation with rotation @ 4C for 2hrs. Pelleted the Protein A/G beads 3300g, 2mins, 4C.
Removed and saved supe (to retain unmethylated DNA). Washed beads with 1mL 1x MeDIP Buffer. Repeated two more times. Saved supe after each wash.
Resuspended beads in 250uL MeDIP Digestion Buffer (50mM Tris-HCl, pH=8.0, 10mM EDTA, pH=8.0, 0.5% SDS). Added 75ug of Proteinase K. Incubated 20hrs @ RT with end-over-end rotation.
Note: The protocol we have says to incubate the Proteinase K digest @ 55C. However, we don’t have a means to do so, since we need a rocker/rotator to keep the agarose beads in suspension. According to various sources, Proteinase K retains >80% of it’s enzymatic activity between 20C-50C. So, I’ve allowed the digest to run longer (24hrs) than recommended (O/N).
After confirming proper fragmentation (~460bp average fragment size) via Bioanalyzer earlier today, began the MeDIP process. Brought fragmented DNA samples up to 350uL with TE. Heated samples @ 95C for 10mins, then incubated on ice 5mins. Added 100uL of 5x MeDIP Buffer (50mM Na2HPO4, 700mM NaCl, 0.25% Triton-X 100), 45uL of TE and 5uL (5ug) of anti-methyl cytidine antibody (Diagenode; 5-mC monoclonal antibody cl. b). Incubated O/N, 4C rotating.
The two gDNA pools (SB and WB) were sonicated using a Covaris S2. Used the guidelines of the manufacturer (listed below) for shearing gDNA to a desired target size (500bp):
Duty Cycle: 5%
Cycels per Burst: 200
Time (seconds): 90
Temp (water bath): 4C
Power Mode: Frequency Sweeping
Sample Volume: 120uL
DNA Mass: ~8ug
Starting Material: >50kb
To be noted, the Covaris guidelines list the use of an “AFA Intensifier” tube, which I did not use (because we don’t have them).
After shearing, ran 250ng of each pool on a 2% TAE agarose gel for fragmentation verification. Also ran 250ng of pre-sonication DNA from each pool as controls.
Lane 1 – Hyperladder I
Lane 2 – R37, Un-sonicated
Lane 3 – R37, sonicated
Lane 4 – R51, Un-sonicated
Lane 5 – R51, sonicated
Sonication with the Covaris did NOT produce the desired fragmentation (500bp smear) in either sample, although the R37 sonicated samples shows a significantly greater degree of fragmentation than the R51 sonicated sample. Not sure how to explain this difference, other than the R51 sample has a greater amount of DNA. Additionally, the results could be explained by the fact that we did not use the AFA Intensifier listed in the Covaris guidelines…
Am consulting with a person in Genome Sciences who has used a Covaris for DNA fragmentation in the past to see if the AFA Intensifiers are indeed necessary and, if so, we can use two of them. Hopefully have an answer soon and be able to proceed with additional fragmentation next week.
8 gDNA samples from SB were pooled and 8 gDNA samples from WB were pooled, using equal amounts of gDNA from each sample (1250ng) for a total of 10ug (see SB/WB Mac’s MeDIP spreadsheet for specific samples/volumes used in pooling). Since samples were stored in pH-adjusted NaOH (see 20100605), they needed to be precipitated in order to have the gDNA suspended in TE for the downstream steps of methylated DNA immunoprecipitation (MeDIP). 10% 3M sodium acetate (pH = 5.2) was added to each tube, then 2.5 vols of 100% EtOH and mixed. Samples were incubated @ -20C for 30mins. DNA was pelleted by spinning 16,000g for 30mins @ 4C. Supe was discarded. Pellets were washed with 1mL 70% EtOH and then pelleted @ 16,000g for 10mins @ 4C. Supe was discarded and gDNA was resuspended in 120uL of TE (pH = 8.0) and spec’d.
The R37 (SB) sample pool yielded 7.056ug after precipitation and the R51 (WB) sample pool yielded 8.834ug after precipitation (started with 10ug). This is good, as 6ug is needed for MeDIP and I wanted to have some (~250ng) available for running as an un-sonicated control on the post-sonication gel. Will transfer 250ng from each pool to separate tubes and then proceed with sonication.