I’ve been sick the past several days, and I’m finally feeling well enough to come in to SAFS. Today, since I’m still not back to normal, I spent the day reading some background information on SRM, Targeted proteomics, and general crab biology. I also outline some goals I have from now until the start of school on September 25th.
SRM, DIA, Skyline reading resources
General Crab Info
Pam lended me a book called “The Biology of Crabs” by G.F. Warner. It’s a great resource for biological and taxonomic background information on all things crustacean.
Goals for before school starts September 25th
- Figure out this Skyline DIA thing once and for all! Emma has been busy, but hopefully next week she’ll be able to look at it
- Analyze the 2015 Oysterseed data
- As soon as the lyophilizer is fixed (hopefully soon!!), test out RNA extraction using Tri-Reagent
- Continue to learn more background info on C. bairdi, Hematodinium, Bitter Crab disease, and the 2015 Oysterseed project
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Today, I finished all Oly larval measurements. The ROI sets are currently on Emu (in the Roberts lab). I will upload them to Github either at the end of this week or when my laptop is repaired.
I also helped dissect oysters for Ronnit’s experiment. We shucked three groups of oysters with each group containing diploids and triploids. They were either dessicated at room temperature or under heat. We took gill, mantle, and abductor tissue for qPCR and collected remaining tissue (excluding the gonad and visceral mass) in a ‘whole body’ sample. All samples are being stored in the first -80 freezer. The whole body tissues are on the final shelf while the other samples are on the 3rd shelf- not in a drawer.
Plan for another protocol test
Time to try this again. At lab meeting, we decided to try to get at least 1 ng/µL of DNA because that’s apparently all I would need for a Qiagen assay. I can’t use the Genome Sciences Thermomixer anymore, so I’m improvising. Steven and Sam suggested that I use a heating block to incubate the tubes at the proper temperature. I would keep the tubes on the heating block for a few minutes, then vortex at maximum speed for a few minutes. I would keep alternating until I kept the tubes on the heating block for at least an hour. I might need to extend the incubation time a bit longer to ensure the proteinkinase K digestion happens properly.
Based on my previous trial, I determined that the Tissue Tearor and two minute vortexing with glass beads provided the most consistently high DNA yield. To recap:
- Tissue Tearor at setting 2 for 20 s (Note: Laura said she used the Tissue Tearor at setting 15 in this lab notebook. She must have used one different than what’s in FSH 213, because there was no setting 15 on the one I used).
- Glass beads + vortexing for 2 minutes
I’m going to use three tissue samples, split between two tubes (each tube corresponds with each method). I don’t want my tube labels to overlap since I’m going to run these samples and the samples from the previous trial on the Bioanalyzer together. Here are what my tube labels will be:
I’m also not going to eliminate RNA from the samples. I would like to do both bisulfite sequencing and RNA-Seq with each sample, so I want to see if the protocol still works.
Kaitlyn’s helping me out tomorrow, so hopefully this will go smoothly! I’d really like to successfully extract DNA that can then be used for bisulfite sequencing :]
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Yesterday (8/28), we set up a 24 hr exposure with triploid and diploid C. Gigas oysters to an elevated temperature + desiccation stress. In total, we had 40 oysters (20 diploid, 20 triploid). 10 control animals were kept for both the diploid and triploid animals in mesh bags in the aquarium in regular seawater. We had to place a foam cover over the mesh so that sea stars wouldn’t get in and potentially interfere with the controls. The other 20 animals (10 diploid, 10 triploid) were transferred to an incubator at 27 degrees Celsius where they were also kept in mesh bags. Exposure began at roughly 12:00 PM. Additionally, we decided to take 2 animals from both the control and the desiccation + elevated temperature exposure at 24 hrs to expose to a 1 hr acute heat shock at 45 degrees Celsius (for a total of 8 oysters exposed to acute heat shock). Thus, our sample size is N = 8 for the diploid/triploid controls and the diploid/triploid desiccation + elevated temperature treatment group.
Originally, we were planning to do a factorial stress response experiment, examining the effects of hypoosmotic stress (low salinity), desiccation + elevated temperature, and the two stressors combined. However, we ultimately decided to go with a simpler experimental design that would be more robust and could give us a baseline stress response to work with. I’m still interested in investigating interactive effects between multiple stressors, especially in triploids, so once we have this baseline single-stressor trial completed, we can definitely think about doing a factorial design!
Today (8/29), we’ll be sampling the oysters at the 24 hr mark, collecting mantle, adductor muscle, and gill tissue for follow-up RT-qPCR. At around noon, we’ll take 2 oysters each from the diploid/triploid control and treated groups to do an acute heat shock at 45 degrees Celsius for 1 hour.
Per this GitHub issue, I’m IDing transposable elements (TEs) in the Crassostrea virginica genome.
I ran RepeatMasker (v4.07) with RepBase-20170127 and RMBlast 2.6.0 four times:
- Species = all
- Species = Crassostrea gigas (Pacific oyster)
- Species = Crassostrea virginica (Eastern oyster)
- Default settings (i.e. no species select – will use human genome).
The idea with running this with four different settings was to get a sense of how the analyses would differ with species specifications.
All runs were performed on roadrunner.
All commands were documented in a Jupyter Notebook (GitHub):
NOTE: RepeatMasker writes the desired output files (*.out, *.cat.gz, and *.gff) to the same directory that the genome is located in! If you conduct multiple runs with the same genome in the same directory, it will overwrite those files, as they are named using the genome assembly filename. Be sure to move files out of the genome directory after each run!
Isadora arranged for different groups of elementary school children from the Refugee Women’s Alliance’s Summer Camp to visit UW today and next week. Today, these children were primarily from Mexico. For our activity, we decided to discuss the many different sources of water in the Puget Sound area. We wanted the kids to feel a connection with their local body of water, and learn some fun facts about the creatures that lived there.
The first thing we did was go around and do introductions. We shared where we and our families were from, and what bodies of water were near us. We then discussed the bodies of water in the area, and the differences between lakes, rivers, and oceans and the animals that live there. Finally, we got to show them the cool creatures that live in the water by looking under a microscope at water from Puget Sound and by going to the fish collection!
Figures 1-2. Marta teaching the campers about the fish collection.
It was my first formal event with younger children, and it was really fun. Everything was cool to them: using a microscope, touching preserved shark specimens, and sharing critter facts. For next week, we’re going to shorten our introductions, incorporate more time for engaging with SCUBA gear, and set up microscopes ahead of time so we know for sure they’ll see things that aren’t just algae. Next week, we’ll have only two Spanish speaker,sa nd the rest will be of Somali heritage. I’m excited to see how they revieve our activity!
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I continued measuring Laura’s Olympia oyster larvae. You can view the measurements here, which is a new file location than previously. I’ve got about 20 groups left and have completed about 30 so far.
Today I finished speed vac-ing (medium heat) the pooled sample. It ended up being too low of volume (14.1ul), so I added 40.9ul of 0.1% DEPC-treated H20. I ran 2ul of the sample on Qubit (RNA HS), and got a reading of 20.4 ul (1,081 ng RNA in the sample)!!!! We FINALLY have a sample to send off for library prep and sequencing! After getting info from NWGC (Chris in the Nickerson Lab at Foege) I put the sample on dry ice and walked it over! It is now in their hands until we get the data back. 🙂
Yesterday wasn’t enough time on the speed vac. So I put it back on (on medium) at 10:10 am. I took it off at 1pm.
I checked the volume of the sample by sucking it all up in a pipet tip (set to 50ul). Then, I decreased the volume on the pipet tip until the liquid was at the tip. It showed that the volume was 14.1 ul. It needs to be at least 50ul. So I sucked the sample back up with the pipet set at 14.1 ul. It all fit.
Then I added 40.9ul of 0.1% DEPC-treated H20 so the final volume was 55ul and vortexed it for 10s.
I then used 2ul of the sample to run on the Qubit (RNA HS) to check the RNA quantity. It read as having 20.4ng/ul of RNA! Meaning that in the sample, we had ~ 1,081 ng of RNA!!! (NWGC requires a minimum of 1000ng of RNA in a sample at least 50ul in volume).
I contacted NWGC to get info on what all they needed before I walked the sample over.
I brought it over on dry ice and handed it to Chris Frazar in the Nickerson Lab in Foege at 2:45pm.
He emailed me and Steven (I cc’ed Sam) to get more info on the sample and what kind of sequencing we want. More information on that to come. They will do the library prep and the sequencing.
I am beyond excited we finally have a sample at NWGC!!
- Try Tri-reagent method of RNA isolation once the lyophilizer is fixed
- Perform qPCR assay to test shellfish primers (GitHub Issue #353)
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Using the program Stringtie for the transcript abundance estimation for libraries from 2 thermal-resistant (TR) and 2 thermal-susceptible (TS) oyster families exposed to oscillatory thermal challenge during 30 days. The program assigned different gene IDs (different from C. gigas gene IDs) specifically in merge step in the output file (/Volumes/toaser/roberto/Hisat_results/stringtie_results/stringtie_merged.gtf) but it has the reference ID (CGI_10000005 for example). As Steven suggested, using finder on stringtie_merged.gtf file, I found the missing CGI gene IDs in the gene expression table (where only 4379 genes had CGI IDs from 60643 total expressed genes).
Doing a test, the stringtie ID “MSTRG.21417” corresponds to DNMT1 gene CGI_10021920 (https://www.uniprot.org/uniprot/K1QQH9). It is differentially expressed between TR (samples Os13, Os14, Os15, Os16, Os17 and Os18) and TS families (Os1, Os2, Os3, Os10, Os11 and Os12) at day 30.
Where phenotype looks with isoform preferences:
As is the case for the isoform #2 (considering up to down in the figure) located in:
scaffold1862: transcript_position: 618219-640790 / gene_id “MSTRG.21417”; transcript_id “MSTRG.21417.4”; ref_gene_id “CGI_10021920” with 34 exons is more present in TR. This could suggest… 🙂