Grace’s Notebook: Speed Vac New Pool of 15 Samples

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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Grace’s Notebook: Gave Pooled Sample to the NWGC for Library Prep and RNA-Seq!

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. 🙂

Sample prep

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!!

Next steps

  • 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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Semi-Annual Progress Report

North Pacific Research Board
Core Research Program
Semi-Annual Progress Report

Project number: 1705

Project title: Effects of temperature change and Hematodinium sp. infection (Bitter Crab Disease) on Tanner crab (Chionoecetes bairdi)

Principal Investigator(s): Dr. Pamela C Jensen & Dr. Steven Roberts

Reporting period: Sept 1, 2017 to July 31, 2018

Submission date: July 31, 2018

 

Accomplishments and results

Sampling
Approximately 400 male Tanner crabs, Chionoecetes bairdi, were collected during the Alaska Department of Fish & Game fall survey and transported to the Alaska Fisheries Science Center (AFSC) Auke Bay Laboratories (ABL) in Juneau. Crabs were placed in insulated tanks at approximately 7°C, the temperature of the bottom water where they were collected. Crab biometric data was recorded, and hemolymph samples drawn for preparation of blood smears and preservation in ethanol. Crabs were held at 7°C for 9 days while ethanol-preserved DNA was extracted & Hematodinium sp. infection status determined for each crab with a conventional PCR assay for Hematodinium sp. (Jensen et al. 2010). On Day 9, based on the PCR results, 180 crabs were equally distributed among 9 tanks (10 Hematodinium-positive and 10 Hematodinium-negative crabs per tank) and the surplus crabs were removed from the tanks. Hemolymph samples were taken from the 180 experimental crabs and preserved in RNAlater Stabilization Solution. After the hemolymph draws on Day 9, over the following 2 days, tank temperatures in 3 ‘cold’ tanks and 3 ‘warm’ tanks were adjusted to the experimental treatment temperatures of 4°C and 10°C, respectively, while 3 tanks remained at ‘ambient’ temperature of 7°C; these temperatures were maintained for the duration of the experiment. On Day 11, hemolymph was drawn and preserved in RNAlater. On Day 27, the final hemolymph samples were drawn and preserved in RNAlater and the experiment terminated. RNAlater-preserved samples were transported to the Roberts’ laboratory at the University of Washington for processing.

tanks

Figure 1. A, experimental treatment tanks at ABL; B, Grace Crandall, the graduate student on the project, taking a hemolymph sample for preservation in RNAlater from a Tanner crab.

 

We anticipated high initial mortality of the crabs due to capture and transport stress, reflected by the number of surplus crabs collected, and indeed, lost ~100 crabs before the experiment began. Throughout the experiment, survival in the cold and ambient treatments was higher than anticipated, with  92% of the crabs in each treatment surviving to the end of the experiment. However, in the warm treatment, mortality was high with 50% of the crabs dying between Days 9 and 11, and all but 1 crab per treatment tank succumbing by the end of the experiment.

 

qPCR assay for Hematodinium infection intensity

In Seattle, DNA was extracted from an aliquot of the Day 27 RNAlater preserved hemolymph. That DNA and DNA extracted from Day 1 ethanol-preserved hemolymph processed in ABL was subjected to a qPCR assay for Hematodinium sp. (Crosson et al., in prep) in order to quantify Hematodinium infection intensity (DNA copy number of Hematodinium). The results will be used to corroborate RNA-Seq analysis results and to investigate the relationship between  infection intensity and gene expression levels in the crabs. The qPCR assay, which is more sensitive than the conventional PCR assay, revealed some Hematodinium positive crabs among the crabs that were negative for Hematodinium via the conventional Hematodinium PCR assay. We are using this additional information to inform our sample selection for transcriptome sequencing and analysis.

 

RNA extraction

Hemolymph samples from 51 crabs (3 sample dates for most crabs, 2 sample dates for some warm treatment crabs: 138 total samples) were processed using a RNA isolation protocol adapted from RNAzol RT. After the completion of the protocol, the isolated RNA was resuspended in 50ul of 0.1% DEPC-treated water, quantified, and stored in -80˚ until sample pooling and/or sequencing. We are currently performing quality assessments on samples for library construction and sequencing that we plan to have underway by the end of this summer.

Based on the timeline in the proposal we are slightly behind schedule in constructing RNA-seq libraries. Due to the high mortality in one treatment group (see below), we decided to evaluate more samples for sequencing than initially planned and to submit samples for sequencing in series, rather than all at the same time. This will allow us to fine tune the sequencing to maximize information from all the crabs, but especially the treatment group that suffered high mortality. We expect to be back on schedule for transcriptome analysis by the end of the year.

 

References

Crosson LM, Wight N, White VC, Vadopalas B, Morado JF, Freidman CS Validation of a real-time PCR assay to detect and quantify Hematodinium sp. in Alaskan Tanner crab, Chionoecetes bairdi.in prep.

Jensen PC, Califf K, Lowe V, Hauser L, Morado JF (2010) Molecular detection of Hematodinium sp. in Northeast Pacific Chionoecetes spp. and evidence of two species in the Northern Hemisphere. Diseases of Aquatic Organisms 89:155-166

 

DecaPod S1E10: Crab Meeting #4

This week we discuss our plan of having three pools sequenced and next steps going forward. Three pools for the current three libraries are as follows:

  1. uninfected from Day 9 (before temperature treatment began)
  2. infected from Day 9 (before temperature treatment began)
  3. “MasterPool” : 10 samples. One sample per each of the following treatments:
    • uninfected, day 12, cold
    • infected, day 12, cold
    • uninfected, day 12, ambient
    • infected, day 12, ambient
    • uninfected, day 12, warm
    • infected, day 12, warm
    • uninfected, day 26, cold
    • infected, day 26, cold
    • uninfected, day 26, ambient
    • infected, day 26, ambient

 

Grace’s Notebook: Notes from Crab Meeting

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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Grace’s Notebook: New Crab Pooling Plan for RNA Seq

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

New plan:
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

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Library #2 – Infected

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MasterPool Library

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New Pool Plan in the big spreadsheet:

Pink: Library 1
Blue: Library 2
Green: MasterPool
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Next Steps

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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