2015 Oysterseed Project:
- Figure out Skyline DIA with Emma (plan for 9/6/18)
- Analyze 2015 Oysterseed data
- Lyophilizer is fixed –> use this along with Tri-Reagent on selected pelleted and supernatant samples
- Continue to learn more background information on C. bairdi, Hematodinium, Bitter Crab disease, etc.
- Practice qPCR to test shellfish primers; learn more about qPCR
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Today I speed vac-ed the new pool of 15 samples for a little over three hours. There is still more than 50ul in the tube, so I’ll put it back in the speed vac as soon as I am in tomorrow morning.
The pooled sample is 150ul (15 samples, each 10ul).
Sam and I went over to the Speed Vac in FSH. At 10:50am, it was started on medium heat.
At 2:10pm, there was still too much liquid. As soon as I am in tomorrow am, I’ll put it back in the speed vac.
Put the sample in (cap open) a slot that has a little bit of paper towel stuffed in the bottom. Close lid. Turn on the machine. Turn the heat to medium. After it runs for a bit, turn on the vacuum (turn yellow valve marked “Vac” toward the machine). Then open up the vacuum by turning the blue dial on top of the machine to “open” (to the right).
To open it later, close the vacuum (blue dial) and then wait til it stops spinning.
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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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Today we had our 4th crab meeting and discussed our short-term sequencing plan for 3 libraries (1: day 9 uninfected; 2: day 9 infected; and 3:a “masterpool” from the reamining 10 treatments). We also discussed our plan going forward with qPCR and creating libraries later and we hopefully will see some cool things with the three current chosen libraries and the qPCR.
I have the meeting recorded, just have to edit and publish it as DecaPod S1E10.
We are going to go ahead with the three libraries proposed in my previous notebook post. Sam is going to check around at the UW CORE facilities available to us. We have to use a UW facility due to budget restrictions (Pam would have to re-negotiate if we wanted to use something else, like Genewiz).
I (with Sam’s assistance and insight) will create the pooled samples such that we will have a tube for each library (3). We will then use the Speed Vac to evaporate off some liquid in order to get a specific concentration (TBD- depends on what the CORE facility requires).
Sequencing takes some time, so while we are waiting for the results to come back, I will compile databases of genomic resources. Namely, find fasta files for those closest related to Chionoecetes bairdi and Hematodinium spp. and create databses. I will also practice using Trinity and BLAST with some geoduck data that we have already so that once the RNAseq data from our crabs comes back, we’ll already have a good idea on how to execute the bioinformatic pipeline.
Once we analyze the data and pick out some genes, we will make primers and use qPCR on individuals. If we see anything that we’d like to look at more closely, we can create new libraries of individuals or pools. I also may extract more RNA since I currently have only extracted RNA from <~50% of the surviving crabs (113 crabs survived the experiment and I have extracted RNA from 51 of them).
I will be learning a lot this summer and I am really excited! Reading and praciticing occasionally doesn’t stick with me as much as actually doing things, so practicing with real data will be very helpful. Pam is also interested in learning more as well, so working with her and potentially teaching her what I learn will further enrich my understanding. Looking forward to it!
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Today I met with Sam and Steven a couple times to figure out what we’re going to discuss this Thursday with Pam. Steven and Sam have come up with three pools that we feel are a good place to start because unfortunately there’s not enough RNA to do the original pooling plan. This post details all the info that I currently have on the plan for our discussion on Thursday.
Links to spreadsheets:
RAW csv (mainsheet): 20180702-crab-sampling-file.csv
Excel spreadhseet (tabs for each pool): 20180702-crab-sampling-file.xls
Start out with 3 pools:
- Pool #1 Uninfected from Day 9
- Pool #2 Infected from Day 9
- MasterPool: The highest RNAng sample from each of the remaining 10 (Pools 3 – 12) pools
This plan will not be able to take temperature into account, but it will allow for gene discovery between the infected and uninfected (Pools 1 and 2). And gene discovery in the MasterPool.
After these pools are sequenced and some targets are ID’ed, we can go in and do qPCR and then plan for some more libraries based on those results.
Below are the details on how I am going to do the pools (very much subject to change as I get input from others):
Library #1 – Uninfected
Library #2 – Infected
New Pool Plan in the big spreadsheet:
Pink: Library 1
Blue: Library 2
If this all looks good, then we can move on to using the Speed Vac and then send them to get sequenced.
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- DIA error rates and move forward
- Pubathon for 2015 Oysterseed paper
- Pick the specific samples to be sent off for sequencing
- Continue reading more papers and background info on crabs in general
- Try out Bioanalyzer again
- Keep posting DecaPod episodes: sample picking for sequencing; background info on crabs in general; brainstorm other topcs
- Decide how many episodes per season
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Because only three crabs exposed to the warm temperature treatmeent survived the experiment, we decided during out meeting on Tuesday (2018-05-22) that I would isolate RNA from warm treatment crabs (infected and uninfected) that made it to the temperature treatment stage (day 12). So today I started that process and isolated RNA from 8 samples (4 crabs, all negative for Hematodinium based on Pam’s qPCR results).
Hemolymph collection data of samples I processed today:
RNA HS Qubit results from those samples (tube number ties back to FRP in the hemolymph data sheet):
Tomorrow I will isolate RNA from two more crabs that are warm temperature treatment and uninfected (6 total uninfected) and 6 crabs that are warm treatment infected (using qPCR data from Pam).
Working on figuring out a way to use R to organize the data into a master file such that each row is an individual crab (as designated by the unique FRP ID number), with columns for each piece of important data we have associated with that crab (hemolymph tube numbers from the sample dates, RNA isoaltion and Qubit data, morphology data, qPCR results). I am enjoying the puzzle-solving-like aspect of this process, but it can be overwhelming sometimes becuase it is a lot of data that is in many different sheets and workbooks. Our master file will include ALL crabs, including those that died after the second sampling date.
Thursday and Friday goals:
- finish isolating RNA
- data organization and master file creation
- data organization and master file creation
- work on and finish a pooling scheme for RNA sequencing using the master file
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