2015 Oysterseeddia Update


layout: post title: 2015 C. gigas oysterseed DIA update date: ‘2018-12-10’ category: 2015Oysterseed tags: [DIA, protocol, Skyline] Today I worked on the DIA protocol and now have an exported report that I think I can start using with MS Stats. Still waiting on some input from Emma, but details are below:

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DIA protocol, and Next Steps

Today I worked on documenting what I did (writing up a protocol of sorts) that is in progress. I am re-doing a couple steps just so that I’m ABSOLUTELY certain where files came from, and I’m putting all the new outputs in a brand-new directory so that it’s just all more organized. I will upload all of the directory and files into a new folder in OWL, which will be linked out in the new protocol. I also got some input on how to set the thresholds that we talked about in the proteomics meeting yesterday, so I’ll try that Monday once my process is fully documented so I can just focus on analysis.

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2015 Oysterseed Plan and Crab Meeting Notes

Crab Meeting today: shared current status of what we’ve got (FISH546 grace-Cbairdi-transcriptome). Additionally, we had a proteomics meeting today and I got some input on next steps for the DIA: set some stringent thresholds, MS Stats, make some tables, etc. I will make a separate post on what I did (protocol) tomorrow as I work with the data. Today’s post will just include a general outline of what I have and next steps.

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Make Taxonomy Pie Charts in R

Today I fiured out how to make pie charts in R, and I did so with the taxonomy output from the BLAST with the C. bairdi transcriptome and the nt taxonomy database. I mostly worked on my repo for Fish546, and it’s still a work in progress. Also, update on crab extractions - Bioanalyzer isn’t reading chips….

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Start new DIA analysis using Walnut, EncyclopeDIA, and Bri-line

Today I tried doing the mprophet model in Skyline Daily. It wasn’t super great and I got stuck, but Emma reminded me that a lot of people who do DIA analysis prefer a new method using Walnut, EncyclopeDIA, and Bri-line. I started this new protocol today and am currently letting Walnut make the new chromatogram library, which will take a while. I’ll move on to the next phase for this tomorrow when the library is done.

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Cold, Infected Pool with RNeasy and DecaPod S1E13

Today I edited and published S1E13 of DecaPod. In this episode I interview Madi Shipley (MS, SAFS) who works with Bering Sea Tanner crabs. I also extracted RNA from 8 samples (all Cold, infected, Day 26) using Qiagen RNeasy and definitely have enough RNA for a pool!

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RNeasy Test Bioanalyzer Results

Today I ran the two samples from Day 26 that I extracted using RNeasy Kit on the Bioanalzyer. The results look pretty good and we can now make a library and see how it goes.

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Submitted GSS poster, and my goals for Th and F

Today at 4:25pm, I submitted my poster to be printed for GSS, which is tomorrow… Steven helped a lot with picking out what information would be interesting to include. My goals for Thursday and Friday include both the Crab Project and the 2015 Oysterseed Project.

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BLASTn with Blue Crab and Prep for GSS Poster

Today I BLASTn -ed Blue crab transcriptome with the assembled C. bairdi transcriptome on Mox. I also finished up some more things in my BLAST to GO GO-slim notebook with uniprot/sprot. Additionally, I got some input from Steven on how best to create a poster for GSS (which is this THURSDAY) so that I can get some cool stuff on there, but not use too many words… which is always a problem I have.

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BLAST on Hummingbird and Transrate

Today I attempted to take my BLAST output on Hummingbird and work through this blast-2-slim notebook of Steven’s. I had gone through this before using Cgigas. I got stuck today at the part where you !join _blast-GO-unfolded.sorted with GO-GOslim.sorted. Will ask tomorrow. Additionally, today I worked on using transrate to look at my C. bairdi assembly using Laura’s notebook as a guide.

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BLAST really running on Mox, 2015 Oysterseed new plan

Today I officially ran BLAST on Mox. It kept failing the past couple days, and Steven pointed out I didn’t actually make the directory I was telling Mox to put the output in, and that I was saying to use blastn when it should have been blastx. I also met with Emma yesterday and spoke with Nick from Skyline to come up with a new plan for the 2015Oysterseed DIA analysis. Also, at lab meeting and Crab Meeting last week, Steven gave me some things to try with the BLAST output and assembled transcriptome for GSS next week. Also, I posted DecaPod S1E12 - Crab Meeting 6. Steven mentioned some things that I should go back and change, so I’ll work on that in my spare time.

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Sent BLAST to Mox, and Plan for rest of Week

I sent a .sh to Mox to perform a BLAST with my assembled C. bairdi transcriptome and swiss prot. I am also working on figuring out how to do this on Hummingbird (on toaster/grace). Additionally, I have come up with a list of things I want to accomplish this week!

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November Goals

Crab Project


RNA extractions and Libraries (Get this done before I go home Dec 21st)

  • Come up with RNA extraction plan for the remaining libraries
  • Perform extractions using Qiagen RNeasy Plus Micro Kit

    Data organization

  • Create new R scripts for adding new Qubit results data to a master file combining hemolymph sampling data and RNA extraction results data

    DecaPod

  • Publish new Crab Meeting episode
  • Record new episode with library plan
  • Record an interview with Maddie Shipley (find a time to meet)
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BLAST with bad Trinity fasta, R plan for adding Qubit data, and Testing out RNeasy Plus Mirco Kit

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.

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Re-run Trinity with correct files

Today I re-ran Trinity because the original files I used that I downloaded to my computer from nightingales were too small and not .fastq.gz. Sam showed me how to do rsync from nightingales to mox. I also am working on doing a BLAST with the finished Trinity.fasta that is too small so that once my real assembled transcriptome is ready, I’ll have a pipeline set up.

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Sent First Trinity Assembly Job to Mox!

Today, with Steven’s guidance, I sent the first job to mox! I am not sure if it will work, but I have it set up to get email notification when the job finishes. Below I provide the link to the .sh script to run Trinity as well as the command line code to run Trinity on mox. I also am starting to work on creating a BLAST pipeline ready. I provide the link to what I’m working on. Currently it’s in a jupyter notebook, but Steven said that this can also be done on Mox, so I’ll work on making it into an .sh script and create GitHub issues to get it checked out by Steven and Sam.

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Worked more on R script for adding Qubit data; Started using Mox

Today I worked more on my R script for adding new Qubit files. Everything works great up until the actual joining of files. After joining, there are extra columns that have the extensions “.x” and “.y”… I think it has something to do with the fact that some columns are factors, some are characters, and some are numeric… I also started using Mox today, but am unsure how to upload the .fastq files from the C bairdi transcriptome data. Waiting to hear back on that in a GitHub issue.

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Crab Project - try RNeasy Kit on some samples; Adding Qubit data to sample file

Today, Steven, Sam, and I discussed where we’re at with the crab samples and RNA extraction. We decided to extract some samples using the RNeasy Mini Plus Kit from Qiagen that Sam has already used and had decent results. In the meantime, I’ll be working on creating a little metadata analysis comparing the different methods used so far, the yields, whether the samples were lyophilized, etc. To do this, I had to figure out how to add the new Qubit data to the crab sampling file. I think I’ve figured out a general work flow in R, and Sam helped me out with some codes to fix some errors I was having.

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RNA extraction and Running .fastq sequence files through FASTQC

Today I extracted RNA from 10 more pelleted hemolymph samples using Tri-Reagent (without lyophilizing). I ran 5 ul on the RNA HS Qubit, and 7/10 had quantifiable RNA. I also started working with the .fastq sequence data from our first sequenced library. I ran the four files through FASTQC and am learning how to determine if the output is good.

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More Tri-reagent Extractions

Today I extracted RNA from 10 samples from Day 26. These still had all three samples in the box, so they are all ###-1 tubes. The QUbit wouldn’t let me download the data from the USB. I’m not sure why, but luckily I wrote down, as I always do, the reads for each tube number. Out of the 10, 7 had quantifiable RNA by the RNA HS Qubit.

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Plan for R and Bioanalyzer results

Today I discussed with Sam about how to use R for making a better master file. We identified some short term and long term goals for me to work on (and get help with via GitHub Issues). I also ran the Bioanalyzer on Test3 (from when lyophilizer was used and using Tri-reagent ), and four samples that I extracted last Friday using (wihtout lyophilizer, with Tri-reagent). The Bioanalyzer didn’t run great because the ladder and the markers didn’t show up… will have to re-run.

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October Goals

Crab Project

  • Identify successful RNA extraction protocol
  • Get extracting RNA going so that we can plan to send some more samples for library prep and sequencing
  • Publish more DecaPod episodes (about 2 - one every two weeks)
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Samples in Lyophilizer got all over the place

At 10:30 am, I went to the lyoppilizer room and met up with Megan. My samples had gotten all over the inside of the manifold. Sam thinks it’s likely because the sampels thawed before they got on the lyophilizer, which is very likely because I brought them over on wet ice as opposed to dry ice. WIll try again at 4:30pm (bringing them over on dry ice) and Sam will grab them in the morning on Thursday. I will also test this out only with screw cap tubes and have them slightly unscrewed. Megan also noticed that the vacuum didn’t get below 100 mtorr, which it should… so the manifold likely wasn’t sealed properly. So Megan added a lot more grease.

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Crab Meeting

Today we had Crab Meeting #5. We discussed the new plan for using the lyophilizer (freeze drier) and Tri-Reagent protocol for extracting RNA. I recorded the meeting and will edit and publish tomorrow as a new DecaPod episode. I also went over to FSH building with Sam and he showed me how the lyophilizer works. On Monday, I will put the samples in and retrieve them on Tuesday, at which point they will be ready for Sam to extract RNA from and for me to run the lyophilized pooled sample on the Qubit.

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September Goals

2015 Oysterseed Project:

  • Figure out Skyline DIA with Emma (plan for 9/6/18)
  • Analyze 2015 Oysterseed data
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Re-tried Skyline DIA

Today I re-tried the Skyline DIA 2015 Oysterseed protocol. Emma has been pretty busy teaching her course these past several weeks and hasn’t been able to get back to me on my GitHub issue (#341). I re-did the protocol using the same files and quadruple checking all the settings. Things still look mediocre. I’ll post the new .zip to today’s file on the GitHub issue so she can take a look at it when she has time.

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Reading and Goals for now until Sept. 25th

I’ve been sick the past several days, and I’m finally feeling well enough to come in to SAFS. Today, since I’m still not back to normal, I spent the day reading some background information on SRM, Targeted proteomics, and general crab biology. I also outline some goals I have from now until the start of school on September 25th.

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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. :)

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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
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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.

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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).

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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…

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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June Goals

2015 Oysterseed

  • DIA error rates and move forward
  • Pubathon for 2015 Oysterseed paper
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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).

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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 LITERATURE ON CRABS, HEMATODINIUM, TRANSCRIPTOMICS AND OTHER “-OMICS”
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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.

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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.

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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:

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Crab Project Updates

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

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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
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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.

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March Goals

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

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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).

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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.

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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.

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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.

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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.

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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:

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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”.

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