Today I worked on trying to figure out what could have made my error rates from last week so awful. I re-did the Skyline DIA protocol many times and ended up realizing that some of my samples only had a few files listed in the protein, while others had all four. Asked Yaamini for clarification. I was told by Emma to make sure that I have four tabs in Skyline. Each tab represents a sample, and each sample has 4 files associated with it.
Today I re-did the Skyline peak-picking error rate for the 2015 Oysterseed DIA and sent off the results to Emma for feedback. I also worked on figuring out how to make the 12 pooled samples that will be sent off for sequencing. Awaiting Steven’s feedback on that.
Tips and tricks for Jekyll notebook posts.
Today I picked samples for sending off to get sequenced. Obviously I have to get this checked with everyone… and I’m guessing they’d rather see it in a spreadhseet form and not color-coded as I do now… have to figure out how to do this.
- DIA error rates and move forward
- Pubathon for 2015 Oysterseed paper
Isolate RNA from warm crabs day 9 and 12 (crabs didn’t make it to day 26). Work more on creating a master file of all data associated with the crab project.
Plan for what I want to get done this week.
Trying out a join in R to figure out what I have in terms of isolated RNA samples.
Images from my trying out the bioanalyzer. And I try again. Neither tries look great. I should try again. Pub-a-thon plan and DecaPod S1E7 published (crab mtg #3).
- Create a visual representation of all of the successfully isolated RNA samples I have and figure out a pooling scheme for next Crab mtg
- Publish new podcast episode every week
- Run bioanalyzer on some samples
- Work on google doc for project
- READ MORE LITERATURE ON CRABS, HEMATODINIUM, TRANSCRIPTOMICS AND OTHER “-OMICS”
Today we had Crab meeting #3 and I tried out the bioanalyzer.
Isolated MORE RNA, Yaamini helps me with organization in R. Plan for tomorrow.
More RNA isolations and plan for data sheet organization for the week. Plan to try out Bioanalyzer.
RNA isolation over weekend to replace those that had “out of range”. Edited and published S1E6 of DecaPod.
More RNA isolation to replace those that had “out of range” RNA qubit results. Ended up contaminating them all, so unusable. Starting my journey of using R for data organization.
Today Pam showed me what she does in excel to move info from one spreadsheet into another based on a shared column. Plan for picking crabs to replace those that had “Out of range” qubit results (either no RNA in sample I pulled out; no hemolymph sampled in first place; or so little that it’s below limit of detection.
Last batch of RNA isolation based on what I picked previously. Working on organizing data sheets to figure out exactly what I have. Plan for this week.
Updates on progress of RNA isolation, DecaPod, and plan for the next week.
So last week was unfortunately not as productive as I had planned as a result of some dog-sitting shenanigans. So, to help myself get back on top of things, I’m making a list of what I want to and will accomplish this week. Firstly, tomorrow I have a presentation in my FISH 511 class, so I will be focusing on that today and tomorrow. The rest of the week will be as follows:
Updates on RNA isolation and Qubit progress and DecaPod progress and plan. Finally post S1E1 on project background!
Result from Qubit of RNA isolation samples and new episode of DecaPod published! Episode on RNA isolation - S1E4.
Updates on RNA isolation progress, DecaPod progress, and Data management plan.
- RNA Isolation
- Isolate RNA from all crabs within the subset I selected (2 more sets of 9 to go)
- Run Qubit on ALL of the isolated RNA samples from the subset I selected
- Edit the three recordings I have that are about:
- RNA isolation
- Crab mtg #2
- Background info on the project (Ep. 1)
- Come out with an episode each week
- Edit the three recordings I have that are about:
- Organize into master spreadsheet - or at least start the process (morph data; qubit; EVERYTHING)
- Add Qubit results to the spreadsheet containing the subset of samples that I’m working with to isolate RNA
More RNA isolation - successful! Plan for tomorrow - Crab mtg #2 and work on DecaPod with Pam.
Today I continue RNA isolation using the newer version of the protocol. Successful day!
Today I use the updated version of the RNA isolation protocol and find that it is so much better and isolating RNA than the generalized protocol. This one is based on what Sam did with extra samples from Sam in the fall of 2017. It adds some extra mixing and centrifuging steps that appear to be the key. Good results.
Today I updated the RNA isolation protocol based on info from Sam, and I made some mistakes in isolating RNA today. Ran Qubit.
Today I finish the error rate determination in Skyline and hope that I did it right so that I can move on with the DIA analysis. I also run Qubit to quanitify the RNA in my samples I extracted from.
I isolate RNA from another batch of 9 samples (3 crabs) and I try to understand what I’m doing in terms of determining peak-picking error rate.
I re-do the DIA analysis from step one. I’m still unsure how to do error rate determination. The protocol is not clear on how to do this.
Get some input from Emma on DIA and I start isolating RNA.
Updates on RNA isolation sample picking; DecaPod; and DIA analysis progress.
I provide updates on status and next steps for my different projects.
My goals for this month are to continue my current work. I also look back on how I did in February.
Today I worked on organization the hemolymph crab data and went to a workshop hosted by UW IT to learn about Audacity for Podcasts.
Today we had our first crab project meeting. Our plan is to meet the first Thursday of the month (subject to change if need be).
Today I began the process of isolating RNA from the “official” crab hemolymph samples, although they can change if I mess things up, because there are many extra samples.
Moving foward in DIA (confusion in determining error rate, so I’m stuck); plans for tomorrow and RNA isolation.
Revisiting what I did last week and my goals for next week: DIA, podcast, RNA isolation.
This quarter I am taking FISH 521, which is the Research Proposal Writing and Professional Development course taught by Julian Olden. This class has been very helpful because it has guided me through the layout of the Crab Project and the plan of how it will all be performed. This is good becuase this is my first quarter of graduate school and I had only heard about the crab project around Thanksgiving of 2017.
Today I tested out Qubit for the first time as well as moving on to step 4 in the DIA protocol.
I tested out the RNA isolation protocol for the first time today, as well as have run into some difficulty in re-doing the DIA protocol.
Updates on RNA isolation; Podcast thoughts and subject matter thoughts; restarting the DIA analysis with the correct files.
Tommorrow I will be trying out RNA isolation on 2 extra Tanner crab hemolymph samples. Try protocol with 2 samples: 465 and 468 Both samples are yellow (from third sampling day) and are from crabs that are infected, immature, and held at ambient temperatures throughout the experiment.
Picking some preliminary samples for RNA isolation and getting questions answered about the RNA isolation protocol.
Notes from a meeting on DIA analysis and the 2015 oysterseed project with Emma, Steven, and Kaitlyn.
Ordering the mics for the Podcast and this week I will test out the RNA isolation protocol.
Testing out the Podcast and how the microphone, garage band, and publishing works.
I would have made this post on the actual first day of February, but I have been sick - the neverending cold. Anyway, for this short month I have some goals I’d like to achieve:
I prep for RNA isolation and understanding the protocol, and continue with the 2015 Oysterseed DIA.
Initial info on making a Podcast from Audacity workshop at UW and Crab Hemolymph samples next steps - testing out the RNA isolation protocol.
Today I chose some crab samples for RNA extraction. I made a chart with the tube numbers for the samples. All crabs are immature males. Due to a massive die-off in the warm temperature treatment groups, we unfortunately only have two infected and one uninfected sample per the final two sample days. As a result, the uninfected samples will not be “pooled”.
New qPCR machine arrived, training with Brian, and preliminary crab hemolymph sample picking for RNA isolation.
I posted the crab data from Pam to Owl, and I hear back from Emma in regards to my error rates.
Finished determining error rate for the DIA analysis and sent my results to Emma. Also, include some background info on the 2015 Oysterseed project.
Update on error rate peak picking - confusion as to how to categorize correct and incorrect picks by Skyline. Includes tips from Laura.
Update on DIA anaylsis of the 2015 Oysterseed project, working on determining the error rate of Skyline’s peak picking.
Dates and links to my old notebook posts on when I spun down the hemolymph samples from Pam.