She is a graduate student (MS) in the School of Aquatic and Fishery Sciences, at the University of Washington. We talk about her thesis work of developing a management strategy evaluation for Tanner crab in the Eastern Bering Sea, her job with the Natural Resources Consultants Incorporated, how she got into the crab world, and her plans after receiving her degree. She also shares some of her at-sea experience with surveying in the Bering Sea.
This week we talk about transcriptome assembly (with our new RNAseq data from NWGC!!) and next steps. Also, we decide that Qiagen RNeasy Kit works pretty well for extracting RNA, and will be used to create the remaining libraries for the project.
Today I ran BLAST with the bad fasta from the Trinity run from last weekend. Will look more at the notebook Steven sent me to do the BLAST stats, goslim, contigs, go slim tables, etc. I also have been getting some input from Sam as to how best to manage adding new Qubit data to a master file consisting of all the hemolymph sampling data joined with the Qubit data results. Finally, I tested out the RNeasy Plus Micro Kit on 4 samples from Day 26, and ran the Qubit.
This week Pam tells us about her recent experience on a survey on the Bering Sea, and we discuss our new plan.
The original RNA extraction method using RNAzol RT resulted in low RNA yields, as well as contaminated samples, likely due to excess salts. We have submitted a pooled sample to the NWGC for library prep and sequencing. The pooled sample consists of samples from day 26 (samples were taken in triplicate) from cold and ambient infected and uninfected treatments. This library will be used for gene discovery.
As we continue investigating better methods for RNA extraction, we will create libraries that will be used for comparison between treatment groups.
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.
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)