1/6/12
Summary: finish dot blot from 1/5/12. Oysters exposed to dietary L-methionine
Procedure:
Results:
external image 20120109-m6i2pdpasqqcd2x35k5njahnge.jpg

The results are inconclusive. The background is really high and there is no clear dilution effect. 60 minutes is the longest exposure time I've done with this assay, but the overall signal was so low (or background was so high) that I didn't feel comfortable stopping the exposure any earlier. If I were to repeat this assay I would probably do a larger range of dilutions (e.g. 1.5 - 0.015 ng) and do slightly longer washes.
1/5/12
Summary: initiate dot blot procedure for analyzing global methylation patterns in oysters exposed to dietary L-methionine (gill tissue)
Procedure:
12/28/11-12/29/11
Summary: isolate gDNA from oysters+L-methionine experiment (see 12/21/11)
Procedure:
Results:
external image 20111230-fsxj1ukywissdpb1e7w7wh7nby.jpg
Next Steps: These samples will be analyzed for global methylation levels using the dot blot procedure.
12/21/11
Summary: A) Nanostring update, B) oysters + L-methionine, sample 48hr time-point

A) Nanostring update: results of 4 C.gigas samples sent 12/8/11 are summarized below

12/20/11
Summary: oysters + L-methionine, sample 24hr time-point
12/19/11
Summary: Start trial with oysters + L-methionine
Procedure:
12/16/11
Summary: General plan for L-methionine exposure. The goal of this small trial is to induce hypermethylation in oysters by exposing them to L-methionine. Background: Administration of L-methionine induces hypermethylation of certain gene promoters in rat brains (Weaver et al 2005). But it should be noted that the effects of dietary L-methionine exposures are not that straight forward (e.g. may cause hypermethylation only in certain genes, in certain tissues at certain times, and may also result in hypomethylation (Waterland 2006)).Oysters will uptake the free amino acid L-methionine in seawater (Nell & Dunkley 1984).
12/14/11
Summary: complete EtOH precipitation of fractions generated using the MethylMiner kit (see 12/13/11). Evaluate separation by PCR
Procedure:
external image 20111214-pp4a4g25jfjdii5d1apd8gus7g.jpg

Results:
I am most interested in the R037 1000mM NaCl Eluate sample, as this is the majority of the methylated fraction of the DNA. The quantification here is likely to be inaccurate since conc. is so low. *I will still estimate recoveries, but probably am overestimating the amount of methylated fractions
Control DNA: >100% total recovery (1ug), 60% was in non-captured fraction, 40% was in methylated fractions (combined). This is generally consistent with expected results (by PCR analysis, not direct quantitation) 70% in super, 30% in captured
Sample DNA: >100% total recovery* (~6ug at the beginning ~7.8ug at the end). Here is the breakdown:
non-captured supernatant: 6.2ug (79% of DNA recovered)
1000mM eluted fraction: 1ug (13% of DNA recovered)
2000mM eluted fraction (this would be the highly methylated fraction): 0.6 ug (8% of recovered DNA)
Evaluation of recovery: The recovery was high overall compared to previous runs (see 11/2010), but the breakdown %wise of each fraction was similar.
Follow-up: PCR was performed to evaluate separation of DNA
PCR for control DNA


Results:
eluate 1 (1000mM NaCl fraction), eluate 2 (2000mM NaCl fraction)
external image 20111214-xuh7bgfya98bphf9r3qammrjn4.jpg
Conclusions/Next Steps: Moving to San Diego, next steps buy plane tickets. Not really. The separation worked, but not as well as it has in previous 2 runs in Nov 2010. Lanes 2 and 7 are positive controls and lanes and 11 are negative controls - as expected. For the left half of the gel, the majority of the non-methylated spike is in the non-captured supe fraction (lane 3), but there was also unmethylated DNA in the 1000mM NaCl elution (lane 4). For the right half of the gel, the majority of the methylated spike was in the methylated fraction (lane 9, and a some in lane 10), but there was also amplification in the in non-captured supe (lane 8). This would NOT be an appropriate separation if the goal was to do a comparative analysis between samples. However, this separation is being used primarily as a means to get a reduced representation of the genome for performing high-throughput bisulfite sequencing, so in this case, I feel the separation is sufficient. The only outstanding issue is that the quantification of the 1000mM NaCl eluate (or methylated fraction) is likely not accurate (conc. too low, no real 'peak' observed at A260). When this sample ships to htSeq they will do QC (size distribution and quantification) on the sample before initiating library prep. If the amount of DNA is not sufficient, I think it would be appropriate to pool this fraction with the remaining fragmented DNA sent to htSeq last week (12/07/11 D)
12/13/11
Summary: complete EtOH precipitation of and perform MethylMiner procedure for R037 pooled oyster sample (initiated 12/12/11).
Procedure:
Next Steps: Complete EtOH precipitation, quant samples and send methylated fraction (1000mM NaCl elucte) to htSeq
12/12/11
Summary: Prepare a pool of C.gigas gill DNA (35 x 51 oysters grown in SB for 10m (sampled 4/10)) to repeat MBD procedure to get sufficient gDNA to prep bisulfite treated library for Illumina sequencing.
12/9/11
Summary: ran bisulfite PCR samples from 12/08/11 on gel and excised bands
loaded 20uL of 1st and 2nd round PCRs (see 12/08/11) on 1.0% agarose EtBr gel
Results:
external image 20111209-8b746cqch3wmbpuxj66863hjxm.jpg
Bands! Bands of the correct size!!!! Ok, in summary: The first round PCR shows a faint band around 800bp as expected. The 2nd round PCRs (left to right, sample, 1st round PCR blank, 2nd round PCR blank), show nice bright band at the expected band sizes (355bp and 547bp). There are faint higher MW bands in both sample lanes. The bright bands were excised and stored immediately at -20 (Mac's bisulfite box). A couple follow up things: 1) looks like the method to determine exon/intron boundarys works pretty nicely (see NB entry 12/6/11) 2) BiSearch software did a nice job w/ primer design.
Next steps: The PCR products are ready for cloning next week.
12/8/11
Summary: A) bisulfite PCR C.gigas Vtg primers, B) EtOH precipitate gonad samples (see rationale for precipitation 12/7/11), C) send samples to Nanostring.

A) Bisulfite PCR of Cg vitellogenin (putative 3'exon See SR notebook 12/1/11)

B) EtOH precipitation of gonad samples originally isolated 7/19/10 and re-spec'd 12/7/11. Samples are P28 and P32 ovary samples from oysters spawned 7/01/10. These samples showed some particulate matter in them 12/7/11 and abnormal spec profiles. Briefly, added 0.1 vols of 3M sodium acetate to 280uL of sample, mixed and then added 2.5 vols of 100% EtOH. Mixed and incubated @ -80C for 1hr. Pelleted DNA 16,000g, 15mins, 4C. Discarded supe. Washed pellet w/ 1mL 70% EtOH (2x washes). Pelleted DNA 16,000g, 5mins, 4C. Discarded supe. Resuspended pellets in a total of 100uL Qiagen Buffer EB (10mM Tris-HCl) and spec'd. Profile is still abnormal. Concentrations (P28: 301.7ng/uL and P32: 223.6 ng/uL ) are likely to be inaccurate.
external image 20111209-fa4ahx4e8ej567tcp6bebje79t.jpg

C) Sent 4 samples to Nanstring for analysis. Based on previous data (see summary 12/7/11), wanted to send a few additional larvae samples and also submitted a sperm and ovary sample. Spec'd all samples 12/7/11, but Nanostring will also get a concentration for these samples prior to running them.
12/7/11
Summary: A) Working summary of Nanostring data, B) quantification of potential gDNA samples for Nanostring, C) EtOH precipitation of 5-aza treated larvae sample, D) Ship MBD sample to UW htSeq

A) Working summary of Nanostring data. Includes a few QC steps (1 and 2), scoring method (3), results (4 and 5) and some interpretation. The original file is in Dropbox>Lab>Mac>nanostring
external image 20111207-myyh2yfbng47bmyuctcqg2bn5k.jpg

B) Re-quant some older gDNA samples. I'm doing this because the sperm sample I sent to Nanostring was much lower than expected based on the original concentration (isolated 7/22/10). I want to make sure I have good samples before they get run in the assay. The idea is to analyze additional larvae samples as well as repeat a sperm sample and include an ovary sample.
Results:
external image 20111208-k5h22ij8ymd8xrngngmq3ksbhb.jpg
Yes, something funny is going on with many of these samples. For example P2, P10 and P28 all had conc.between 100 - 300ng/uL in July 2010 (7/22/10). There were some particulates in some of these samples, I wondered if some of the DNA had precipitated out? so I gave them a good spin and re-quanted (see 2nd reads of P28 and P2), it appeared that the conc. had gone up for these second reads, but the spec profiles did not show a nice peak at 260 so I'm not sure what's going on here. I would like to try to EtOH precipitate these samples again, probably in a Tris buffer (instead of 8M NaOH pH adjusted w/ HEPES which is the DNAzol buffer).
Next Steps: I need to EtOH precipitate the 5aza tx larvae sample to get it to a higher conc. for Nanostring before I send it. There is a 'control' sperm sample that looks good for analysis (P19), the ovary sample that had a high concentration and good A260/A230 (P17) was a vinclozolin treated oyster. It could be worth analyzing this sample, but will probably want to try to EtOH precipitate the other ovary samples to get a good control.

C) EtOH precipitation of 5-aza treated larvae. Briefly, added 0.1 vols of 3M sodium acetate to 400uL of sample, mixed and then added 2 vols of 100% EtOH. Mixed and incubated @ -80C for 1hr. Pelleted DNA 16,000g, 15mins, 4C. Discarded supe. Washed pellet w/ 1mL 70% EtOH. Pelleted DNA 16,000g, 5mins, 4C. Discarded supe. Resuspended pellets in a total of 100uL Qiagen Buffer EB (10mM Tris-HCl) and spec'd.
external image 20111208-gb7ujebu98hgnub12yfm6x2kes.jpg

D) Send methylation enriched gDNA to htSeq for Illumina Whole Methylome sequencing. This enriched sample was generated using the MethylMiner kit (Invitrogen). The sample is a pool of gill tissue from 35x51 oysters grown in Samish Bay (R037) for 10 months (see 11/12/10 - 11/15/10 for prep). Specifically, the 1000uM NaCl eluted fraction (methylated) is being sequenced. This same fraction was also directly sequenced (no bisulfite conversion) in March of this year (see 3/7/11 for this sample prep). I spec'd the sample (NOTE: I spec'd this sample in section A but used the wrong buffer to blank. I re-spec'd w/ TE buffer): concentration 15.2ng/ul, 260/280: 1.9, 260/230: 1.1. A total of 0.5ug was shipped on ice, O/N to UW htSeq.
12/6/11
Summary: designed bisulfite PCR primers for the last ~840 bp of Cg_Vtg (AB084783)
Procedure:
external image 20111206-xieah2485ehsku18kw96symnbt.jpg
11/30/11
Summary: 1) ran bisulfite PCR samples from 11/29/11, 2) completed C. gigas larvae DNA isolations initiated 11/29/11.
Bisulfite PCR:
loaded 20uL of 1st and 2nd round PCRs (see 11/29/11) on 0.8% agarose EtBr gel
Results: no bands were observed for either the first or second round PCR for both genes (estrogen receptor or vitellogenin)
Interpretation: This is not really that unexpected. The primers were designed from an mRNA sequence. It is likely that the gDNA contained introns (first round PCR was to amplify ~1500bp of each gene, the second round to amplify ~700bp of the 1st round template), and so it is possible that band sizes were too large to amplify under these conditions. Alternatively, it is possible that primers were designed across an exon/intron boundary and therefore did not bind to the genomic sequence.

gDNA isolation:
Results:
external image 20111201-e681bd2m6wpmitfy8mkfdijjcy.jpg

Interpretation: Isolations did not go so well. Only 2 of the samples (bold above) had decent recoveries. There are a few reasons why the recoveries were poor. 1) possibly did not have enough larvae for some of the samples (see observations above), 2) larvae have tiny calcified shells, may need to crush larvae or do a more extensive pK digestions to recover DNA from a small # of larvae.

11/29/11
Summary: 1) PCR of bisulfite treated C.gigas gDNA - ER and Vtg gene. 2) Initiated DNA isolation of larval C.gigas samples
Procedure Bisulfite PCR:

Procedure gDNA isolation:
11/1/11-11/2/11
Summary: isolate gDNA from 6 oysters (C.gigas) exposed to 100ng/L EE2 for 96hr (see 10/27/11 for experiment). Samples to be used for Nanostring study. Also included an 'alive' diploid oyster exposed to low pH (sample from integrative environmental physiology A1D)
Procedure:
Results:
external image 20111102-mt2cp2yb44jd3djh1bs2a4si44.jpg

10/31/11
Summary: complete 96 hr EE2 experiment initiated 10/27/11
Procedure:
Sampling: Measured all oysters (data here) Sampled gill, digestive gland and mantle from each oyster 18 total (6 per treatment: control, 50ng/L EE2, 100ng/L EE2). Samples were stored on dry ice during sampling then moved to -80 freezer marked EE2 C.gigas MG 10/31/11. For 100ng/L oysters, took an additional gill sample and stored at -20. Will initiate DNA isolation for nanostring tomorrow with these samples.
10/27/11
Summary: begin experiment dosing oysters with 17alpha-ethynylestradiol (EE2) at 100ng/L and 50ng/L (total duration of exposure to be 96 hours)
Procedure:
Next Step: Perform a complete water/treatment change on Saturday the 29th.
10/25/11-10/26/11
Summary: cont. from 10/24/11. Select colonies for purification, grow O/N in liquid broth, purify plasmids
Procedure:
Next Steps: These are not high priority samples, I will just put them on next sequencing plate that goes out. I will also submit the PCR products for direct sequencing as well. Literature/interwebs always says that bisulfite treated products 'need' to be subcloned, not sure why. Talked to 2 people at ESF conference - one said they do direct sequencing all the time, the other said direct sequencing never worked for them. Sounds like we should try both.
10/24/11
Summary: cont. from 10/20/11. Today: Repeat PCR-screen for insert using 1 vector primer and 1 gene specific primer
Procedure:
Results:
(HyperladderII used: top bright band-2000bp, middle - 1000bp, lower-300bp)
external image 20111025-rfup14t53qsfyfk1cqrm9immh1.jpg
Summary: Yeah, still not sure what I cloned. Bands were not observed for the master mixes using the M13 reverse primers (lower half of gel (not labeled)) indicating that the inserts (what ever they are) were inserted in the 5' ->3' direction. For the PCR's using the M13 (F) primer and gene specific reverse primers multiple faint bands were observed for a majority of all 3 PCR products. The expected band size is ~1000bp for all 3 products. There is a faint band at 1000 bp for a majority of the lanes, but there are also higher MW bands as well. Some lanes show bright bands around 200bp. Since 1 insert specific primer was used, empty vector should not amplify. All in all, results are a little confusing still. In order to determine what has been cloned I will continue on with plasmid preps for 4 of the clones for each PCR product.
10/20/11
Summary: cont. from 10/19/11. Today: Repeat PCR-screen for insert using vector primers
Procedure:
Results:
external image 20111021-bja1u2iu9a9w9ccf14anxe5hb1.jpg
Conclusions/Next Steps: The expected band size for the insert + amplified bit of vector using M13 primers would be around 1000bp for all 3 of the PCR products. None of the colonies had 1000bp bands. Empty plasmids would be around 200 bp, so it looks like there is some kind of an insert for some of the colonies (bands >300bp). I am stumped about the band >2000bp. In order to see if the desired product (or part of the desired product?) has been cloned, will re-screen colonies using 1 vector primer and 1 gene specific primer.
10/19/11
Summary: cont. from 10/18/11. Today: select colonies and PCR-screen for insert using gene specific primers
Procedure:
Results/Next Steps: No bands were present for any of the colonies selected. Also included a 'positive control' by loading 2uL of the extracted band for each template - no bands visible for these either. Results are inconclusive as positive controls did not amplify. Repeat PCR, try using vector primers (M13) instead.
10/18/11
Summary: initiate cloning of PCR products from 10/13/11 (bisulfite treated Cg_hsc70) using TOPO TA Cloning Kit
Procedure:
10/17/11
Summary: run PCR products from 10/13/11 (bisulfite treated Cg_hsc70) on gel and cut out bands
Procedure:
Results:
external image 20111018-kx6qr8qtgja6cm39jcnhqr3914.jpg
negative controls were negative. PCR 1 gave very faint band ~650 bp, PCR 4 had bright band @ 700bp and PCR 5 gave mid-intensity band @ 800bp (these bands are the same size are previous bands (from March 2011) - The expected band sizes for all 3 are between 600 and 700bp.

Next Step: clone PCR products
10/13/11
Summary: PCR of bisulfite treated C.gigas gDNA - hsc70 gene. I am repeating what was performed back in March 2011 with a subset of the nested primer pairs because the products didn't clone the first time around. Changes from March: anneal temp dropped from 58C to 55C (58C was to high for product 'A' primers), added 2uL of template to 2nd round PCR instead of 1uL.
Procedure:

10/06/11
Summary: 3'/5' RACE PCR C.gigas DNMT1 cont. from 10/5/11. Nested PCR and visualization of PCR products
Procedure:
Results: Results were similar to those performed using 5' RACE ready DNA on 8/3/11. No bands were observed for the original PCR or the nested PCR. Negative controls were negative.
Next Steps: Send the 800bp band from the 3'RACE for sequencing (early next week?). I will combine this sequence with the 1366bp already sequenced. I may be able to get additional sequence bioinformatically using some of the newly released C.gigas sequencing data.