Yesterday, I went with Laura to Manchester to help clean and de-symbiont about 1600 oysters. There were 7 or 8 of us working, so we got it all done in a little over three hours! And, it was thankfully a beautiful, sunny (cold) day. I scraped off tons of barnacles, limpets, algae, and red worms.
Today, I worked more on the DIA analyses protocol. Specifically, Step 2. Some issues came up, so Steven and Sam have both been helping address them so that we can move forward. I am now looking at 3a and 3b. My task is to work on understanding what these steps mean, and then Steven said we’ll wait and see what we can do about running Pecan. Apparently, it’s difficult to use, and may have to be sent to Emma to run.
I then revisited this GitHub Issue and took some images to provide an example of what this project would be like if I followed suit with what I’ve done in the past.
Sam, Steven, and I (I mostly stood watching) worked on connecting the titrator to the computer (Swan). It was complicated, but I think they got it to work. My task for tomorrow is to make the NaOH (0.1 mol/L) solution in the titrant bottle so that we can hopefully start moving forward with the titrator!
(1) 2015 OysterSeed Data
Today I revisited the 2015 OysterSeed skyline project. I left off at Step 5b .
I am going through ~100 peptides to check that Skyline chose the correct peaks. Peaks are there when peptide or transitions were detectable, and the greater the peak, the stronger the detection was.
I am not sure what to do when the peaks are inconsistent across all oyster seed samples within a peptide. I believe that in Yaamini’s SRM protocol, I was to delete the peptide if the pattern was inconsistent between samples.
Additionally, I am supposed to determine the per-sample and per-peptide error rates. To do this, I will be following what Yaamini did in her spreadsheet: here. Unfortunately, I asked about this after already checking 26 peptides. I didn’t keep track of which ones I checked, so I’ll have to start over tomorrow. I don’t think it should take too long.
(2) Roberts Lab Onboarding
I am going to transition to using a GitHub notebook in order to keep things to do with my projects more organized.
Today and yesterday I finished spinning down, and saving the supernatant and pellet of the final samples.
I added the labeled boxes to the -80 (Tray 14).
Pam picked up the 50µl samples this morning.
Today I was in for a little over an hour labeling tubes in preparation for tomorrow and Wednesday. I will be spinning down the final batch of Tanner Crab hemolymph and RNAlater mixtures, and saving the supernatant and pellets in separate tubes in the -80.
Today I set aside 50µl of crab hemolymph and RNAlater mixture for Pam. 117 tubes for the 117 crabs that remained at the end of the project. Storing them in labeled boxes in the refrigerator in Rm 213.
Next week I will spin down all the tubes, keep the supernatant and pellets in separate tubes and store in -80. Hoping that more P1000 pipet tips will arrive by then to help speed things up.
(1) 2015 oyster seed data
I went through some more peptides on SkylineDaily with the 2015 oyster seed data. Protocol calls for checking ~100 peptides to ensure correct peak was selected (Step 5).
(2) Crab project
I met with Steven and Pam today to discuss the overall project and next steps.
Pam brought the most recent samples. There are three samples per crab, and 6 per warm-water treatment crabs (because only 3 survived). Total of 117 crabs.
I will save 50µl of hemolymph/RNAlater mixture for Pam from each crab (117 tubes total). Then I will spin down all the samples (114×3 + 3×6 = 360 samples total), remove and save the supernatant, and place supernatant and pellet tubes in -80.
In January, we will start processing the RNA and pooling the samples to be sent off for sequencing.
Crab hemolymph samples:
Pink-capped were sampled 11/10/2017
I spun down the hemolymph samples (5000g for 10min) and saved the supernatant and pellets in separate tubes. The boxes were placed in the -80 (tray 14).