New Hemolymp sample and Qubit file; New RNA-seq short-term plan

Today Sam, Steven, and I met and decided to pool the 15 samples that had quantifiable RNA (Qubit RNA HS results) from the 40 samples that Sam processed using the Qiagen RNEasy Mini Plus Kit into one sample to be sequenced. Also, they both helped me learn more about data management and creating a better file that contains the crab hemolymph sampling data and the Qubit results data.

Read More

Streamlining Processing of Adding Qubit data to Master file

Today I played around with GitHub and R Projects. I’ve figured out a way of making adding Qubit results to my hemolymph and Qubit results master file more easy. It isn’t perfect and there are likely some things that I don’t know about that could make it easier, but I’ve detailed the new method below. I also noticed today that the tube_number column in what I did yesterday got all messed up with the merge in R. Detailed below.

Read More

Crab RNA-Seq thoughts; Merging columns in R

Yesterday I shared some preliminary thoughts (in a GitHub issue and detailed below) on what we can do for our first Crab RNA-Seq library. Today I learned - through a whole mess of comments on a GitHub Issue because my stuff isn’t organized well - how to merge data in columns that are the same in R, so that you don’t have duplicates (example: “Test_Date.x” and “Test_Date.y”). Steven mentioned I should go over with him how to name files… which sounds good to me because I think I’m taking too much time out of my day renaming and replacing files as I update them. Sam also mentioned that my R Project is difficult to work with because it includes files that belong to two different repositories. So I’ll work on cleaning that up and making it easier for future collaboration and help.

Read More

New Plan - Lyophilizer; DIA Github Issue

Today I met with Steven and Sam to make a new plan for the crab RNA problem. We’re going to use the lyophilizer (freeze drying machine that goes below -80˚C) on 8 new pelleted hemolymph samples from Day 26; 8 supernatant samples that correspond to those 8 pelleted; and a pooled sample of all the ones Sam processed using the Qiagen RNeasy Kit that had “out of range” Qubit results. This will have to be post-poned for a bit becuase the lyophilizer is currently being fixed… waiting for an ETA on that. In regards to Skyline, I re-visited what I did before I left for vacation and went through all the files used and settings chosen. I put all of the information in a new GitHub issue per Steven’s request.

Read More

Learning code to change Factors to numbers

Today I noticed that the code Yaamini showed me yesterday to change the “tube_number” column values in the 20180522-all-crabs-hemo.csv from Factor to numeric ended up changing the tube numbers, which meant that the spreadsheet I made yesterday was wrong. So, Sam taught me how to make the “tube_number” column from Factor to numerics without changing the value, as described below.

Read More

Hemolymph sampling and Qubit data spreadsheet; Samples for Sam to run RNeasy Mini Plus Kit

I am picking some samples for Sam to use an RNeasy Mini Plus Kit on. They are from Day 26 (samples were taken in triplicate), and 10 were picked from each of the following groups: uninfected, cold; infected, cold; uninfected, ambient; infected, ambient. I also made a spreadsheet that contains the hemolymph sampling data of all the crabs in the whole experiment (including those that died) left_join-ed with Qubit results data. However, after going through my Qubit csv folder, I found a couple of .csv’s that didn’t make it into the consolidated qubit csv. So I am currently trying to join the hemolymph data csv with the UPDATED Qubit results data based on tube_number, but am getting an error.

Read More

August Goals

Crab Project:

  • Continue working with Sam and Steven to find solutions for the RNA isolation issues I had
  • Finish creating a good, readable spreadsheet of all the crab data
Read More

My Bioanalyzer and Sam's RNA Clean-up Kit and RNeasy Plus Mini Kit results

Today I ran the Bioanalyzer on the samples Sam isolated RNA from months ago using RNAzol RT as well as some of the RNA isolated from samples that I have done. The results were not good - no dye showed up, so maybe I did it wrong. TBD. Also, Sam used a kit to clean up the RNA that I have isolated and then ran Qubit and Nanodrop1000, as well as used RNeasy Plus Mini Kit to isolate RNA from untouched hemolymph samples. Low concentrations of RNA, but at least there is RNA. The results from the RNeasy Plus Mini Kit isolations are clean and the RNA on Qubit and Nanodrop1000 are good.

Read More

Trying out RNA protocol with 6x original protocol's volumes of reagents

Today Steven, Sam, and I met to talk about the issue with poor results on the QUbit and Nanodrop1000 on the pooled hemolymph samples I prepared. Short-term plan is to try out the RNA protocol, but multiply all reagents times 6 (so, use 6ml of RNAzol, etc.) to see if that ratio would help; run the Bioanalyzer on the samples that Sam processed as well as some random samples that I isolated. Today, I did the 6x RNA protocol on 4 of the extra samples from day 26 (samples from day 26 were taken in triplicate).

Read More

Skyline DIA with new BLIB made using Walnut

Today Walnut finished up using Walnut to make a new BLIB file with the 2015 C.gigas Oysterseed raw files. Then, I used the new BLIB in SKyline, along with changing the minutes to 5, and double-checking I used the same .mzML files used in Walnut as my results in Skyline. The peaks still look pretty bad. I just emailed Emma…

Read More

Trying out Walnut; Crab Pool Update

Today I started using Walnut (upgraded PECAN) to create a new BLIB file for the 2015 Oysterseed project. Hopefully this will improve the error rates in Skyline. Also, I called Pam to work on the NPRB progress report. Additionally, I detail Sam’s updates on the status of the Crab pool samples from his notebook post. The RNA needs some cleaning so he suggests trying RNeasy Cleanup Kit. I will wait until he returns Monday and speak in person with him and Steven to figure out what to do next.

Read More

Crab Pools and Skyline Update

Today Sam speed vac-ed the pools and put them on ice. We mixed the samples and ran Qubit. The readings were way too low. Sam got out hte Nanodrop and we ran them on that… and the readings were bad. Since I am out of town the rest of this week and half of next, Sam is going to try to fix some things, detailed below. In terms of Skyline, Emma has been in contact with Nick today and others from the Skyline crew. She is working on figuring out what the issue is with my super high error rates. At the end of today’s post, I also outline rest of July goals and summer goals.

Read More

Speed Vac Pt. 1 + Testing Advancing Peak Picking in Skyline

CRAB RNA POOLS: Today I started using the Speed Vac on the three pools with Sam. They were running on low temperature from 10:30am to 1:15pm. Not much liquid had evaporated. From 1:15pm-3:15pm they were run on medium temperature. Still not enough liquid has evaporated, so Sam will put them in the Speed Vac when he gets to FTR tomorrow morning. SKYLINE: I tested out the tutorial that the people from Skyline suggested I try out. Not sure how it turned out honestly, but Emma said she can take a look at it with me tomorrow.

Read More

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.

Read More

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.

Read More

Reading up on Trinity and Interviewed Genevieve Johnson for DecaPod

Today I did more reading on how to start assembling a transcriptome using Trinity with plans to formulate the beginning steps tomorrow and I interviewed Genevieve Johnson (University of Alaska, Fairbanks) on her thesis project doing Tanner Crab population genetics for a new soon-to-be published episode of DecaPod.

Read More

Update on Crab Pooling and Plan for next week-ish

Today I met with Steven to talk briefly about the crab sample pooling (need more info on how to proceed- will wait for Sam’s return in 1.5 weeks), and about things I can do in the interim while the samples are being sequenced: practicing Trinity with Geoduck transcriptome data; DecaPod; 2015 Oysterseed project.

Read More

2015 Oysterseed Skyline questions

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.

Read More

Samples for Sequencing

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.

Read More

June Goals

2015 Oysterseed

  • DIA error rates and move forward
  • Pubathon for 2015 Oysterseed paper
Read More

Bioanalyzer and Pubathon

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).

Read More

May Goals

Crab Project

  • 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

RNA isolation and Data org

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.

Read More

Data Organization- info from Pam in excel

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.

Read More

Weekly goals

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:

Read More

Crab Project Updates

Updates on RNA isolation and Qubit progress and DecaPod progress and plan. Finally post S1E1 on project background!

Read More

April Goals

  • 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
  • Podcast
    • 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
  • Data
    • 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
Read More

RNA iso with new protocol

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.

Read More

March Goals

My goals for this month are to continue my current work. I also look back on how I did in February.

Read More

Crab Meeting

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).

Read More

RNA isolation

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.

Read More

Moving forward with DIA and plan for RNA isolation

I figured out how to upload results in Skyline such that there are 4 tabs (because I have 4 samples) and each tab has four files associated with it. Moving foward in DIA (confusion in determining error rate, so I’m stuck); plans for tomorrow and RNA isolation.

Read More

Thesis Proposal Writing

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.

Read More

RNA isolation protocol and plan for tomorrow

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.

Read More

February Goals

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:

Read More

Preliminary Crab Sample Selection for Seq

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”.

Read More