Today I shipped samples to Genewiz for individual RNAseq. Details in post.
- Extract RNA from all remaining samples (Sam and Kaitlyn are going to keep helping, I think)
- Prepare for qPCR on individual crab samples
- Run qPCRs
- Start working on crab paper
Today I submitted 6 pooled samples of Crab RNA to NWGC for QC. After they run QC, they’ll let us know what our sequencing options are.
Today I extracted RNA from 24 samples. Day 9, 12 infected and 12 uninfected. I did not run them on the qubit yet. Will do later this week.
Today I tried to concentrate the 6 pooled crab RNA samples. Long story short, a lot of RNA was lost in the process and the “concetrated” samples are now well below the requirements for NWGC for RNAseq. Details in post. Next steps, I’m going to extract more RNA from samples from Day 9 so we can bulk those pools up if we want to try to send them to NWGC still. Then I’ll extract more RNA from samples that will bulk up the other pools, too.
Received the Zymo RNA Clean and Concentrator Kit -5 yesterday. Plan is to concentrate my 6 pooled samples. I tested it out on some test pools I made quickly. Short answer: unclear if it worked because the initial qubit RNA HS readings of the test pools were both “TOO LOW”… and after concentrating, one test pool was still “TOO LOW”, while the other was 2.20 ng/ ul in a 2 ul sample from RNA eluted in 35ul. Details in post. (Also at the end of the post: general updates on crab project and my January plans).
- Concentrate the 6 new pooled crab samples (recieved that sample kit of the RNA Clean and Concentrator -5 kit from Zymo – have a lot of time tomorrow to work on this)
- Submit 6 new pooled samples (to NWGC because we have the quote from them)
- Work on new results for AMSS presentation (my talk needs to be done by Monday, January 27th)
So, it turns out my 6 pooled samples aren’t concentrated enough for NWGC to sequence. They are all at around 25ng/ul, but they need to be at least 50ng/ul. I called zymo yesterday to find out how best to do that. They recommended a kit (free sample coming next week). With Sam’s guidance, I tried using the Microprep Kit columns and washes to concentrate some pools that I made today out of extra extracted RNA. It kinda worked, I think… but there was some loss of RNA, so I’m worried about using that method with my pooled 6 samples. Details in the post.
- Submit 6 new pooled samples to NWGC (waiting to hear back about how to submit samples (GitHub Issue: #798))
- Work on assembling transcriptomes and moving forward with other 4 pooled libraries we currently have
Today I pooled samples for the 6 new libraries for sequencing. There is over 1000ng in each pooled sample, so everything is ready to go! Details in post.
I extracted RNA from the same tubes that I worked with a lot the past couple of weeks on the bioanalyzer and Nanodrop and qubit because there ended up being not much left. RNA was in all samples. Details of extraction and next steps in post.
Today, I continued to prepare a presentation for GSS 2019 (this Thursday - my talk is at 1:30pm). I met with Steven and discussed a plan for results, and he shared with me the analyses he’s done during the past couple of days. Details of results and what I’ve done in the post. I also tried doing edgeR stuff yesterday and didn’t get super far, but details will be at the end of the post for my future self (won’t do edgeR for GSS).
Today I went through the DNase-free plan with 4 of the extracted crab samples. There was initially quite a bit of DNA, then after I used the Turbo DNA-free Kit, there was significantly less DNA. The Bioanalyzer still didn’t look super great… detail in post. Also, I’m working through kallisto and have abundance files - working to figure out next steps in order to hopefully create a heatmap of differential expression between the infected and uninfected crabs (combining day 12 and day 26). Details in post.
Today we came up with a plan to address the issues that came up with the weird bioanalyzer results from yesterday (GitHub Issue #792). I also got some help with kallisto and am working on getting some analysis done for looking at differential expression (hopefully have some new results for GSS talk next week).
Today I tried doing several things, none of them super successfully. First, I tried using Kallisto to get some quick differential expression results from the new bairdi RNA-seq data. It didn’t work, but I just need to look a little more into it. I didn’t spend too much time on it. Secondly, I tried running 11 of the extracted RNA samples on the Bioanalyzer - things do not look good. Thirdly, I ran those same samples on the NanoDop. I am not sure what to make of the Nanodop results yet.
Today, Sam, Steven and I met to discuss a pooling plan for libraries (details in post). From that meeting it was decided that I should extract RNA from more day 12 samples, specfically from cold, warm, and infected and uninfected treatments. All 24 samples had RNA. An updated sample summary is provided at the end of the post.
This coming week:
- Finish extracting RNA for pools by next week
- Select samples for 6 pools
- Day 9 infected
- Day 9 uninfected
- Day 12, cold, infected
- Day 12, cold, uninfected
- Day 12, warm, infected
- Day 12, warm, uninfected
- Run those samples for pooling on bioanalyzer; if they look good, pool samples
- Have 6 pooled samples ready to send over to NWGC by end of next week
Today I extracted RNA from 24 more samples from Day 12. Mix of infection status and temperature exposures. There was RNA in 23 out of 24 samples. No obvious mistakes were made, and at the end of the post I provide an updated RNA sample summary in preparation for our crab RNA meeting tomorrow.
Today I did some more extractions, but moved on to day 12. I did a mix of all groupings (temperature and infection statuses). All extracted samples had detectable RNA! Details in post. Additionally, I’ll give a summary table of what all I have extracted so far.
Today I did another round of Day 9 infected (12) and uninfected (12) tanner crab hemolymph RNA extractions. I also ran 2ul of the eluted 15ul RNA on Qubit. Results and details in post.
Today I ran 2ul of the 24 samples I extracted yesterday. 20 out of 24 had RNA. Details in post.
This post contains my notes on the Zymo Research Protocol for Quick-DNA/RNA Microprep kit, and Sam’s notebook post to create a protocol for extracting RNA. I also include my extraction plan for this next week as I try to get enough RNA to get two more libraries (Day 9 infected (1), and Day 9 uninfected (2)).
Oysterseed DIA - trying to characterize list of detected proteins in relation to _C. gigas_ proteome
I’m still working on wrapping up the results and discussion of this paper. Steven recommended I look into characterizing the list of detected proteins (2,808) in relation to the whole C. gigas proteome. Detailed in post:
I’ve been working on finishing up the Oysterseed DIA paper, and met with Steven today to discuss it. Details in post below:
In this post I will detail what I did for PCSGA 2019. I did some new analyses and found some new results from our first assembled transcriptome.
Today I officially got an email that the RAW oysterseed data and *.elib search files have successfully been uploaded and published on PRIDE. I have some plans for finishing up this oysterseed project by October 20th, details in post.
- Keep tabs on data - when it arrives, begin trimming, assembling, annotating process
- Prepare for PCSGA: take some of what I presented for EIMD 2019, and add more about the research project
- Interview Genevieve and Shanelle for DecaPod episodes
I’ve been working on finally wrapping up the 2015 Oysterseed DIA project. I feel like I’ve tied off the paper fairly well this past week, and have been working on cleaning up and organizing the repository today. Details in post.
The past three or so days I’ve been meticulously searching through all of my notebook posts and RNA isolation result files to create a master table of all of the hemolymph tubes that have been processed both by myself and Sam. Additionally, I made a note as to whether all of the sample was used, or if only some of the slurry was used (meaning that there would still be some in the -80). I’m pretty sure that I have a complete list now, but will revisit in a couple days to give my eyes a rest! Details in post.
- create better system for keeping track of samples
- prepare for when seq data arrives: revisit reads trimming, transcriptome assembly, annotation, etc.
- start thinking about/preparing for qPCR portion of project
- update bittercrab.science with crab-related posts from my .io notebook
- interview Genevieve Johnson for 2 episodes: her thesis results (tanner crab pop. gen.); transcriptome assembly process (her new PhD work)
- interview Shanelle (her PhD project outline, goals, etc.)
- ID conferences to attend
- prepare abstracts and titles to submit
Today in Hackweek, we went over what I could do to have a better hold on my crab data sheets… details in post
Today we had our first crab meeting in MONTHS. Not much has changed due to the fact that we’re still waiting for our sequence data, but we discussed some things to do in the interim, met Shanelle and talked about her project and what she’ll be doing in our lab, and also talked about how I should start looking into conferences to present the research at!
I am currently taking the Ecology of Infectious Marine Disease course at UW Friday Harbor Labs until July 19th. It’s been super fun and has really made me even more excited to be in this field and studying disease!
Today I sampled the buckets for D-hinge development. Overall numbers were low, considering that we put 300,000 eggs in each bucket. The best group was treatment group 2: no KCl exposure, but fertilized. Details in post:
Today was Day 1 of the second trial of geoduck stripspawn. We created an experimental design that takes into account both KCl dosage, and duration of the dose exposure. Overall things went pretty well (details in post), and on Wednesday, I’ll check for D-hinge development.
Today we took a look under the scope of the eggs in the silos from Friday’s egg development trial (Notebook post).
Today I looked into trying to figure out why we saw polar body development as early as we did last week during the first trial of KCl-dosage experiments (Day1, Day2). Details of the experiment are below, but there was egg development in the negative control (no sperm, no KCl), so I’m not sure what this all means.
I leave for FHL on June 16th for a 5-week intensive course on the Ecology of Infectious Marine Disease!! Lots to do before I go.
Today we checked to see what development occurred since Wednesday’s strip spawn and KCl-treatment trials. There were so few eggs in the tripours to begin with, that there ended up being way too small of numbers of D-hinge larvae that I ended up dumping the trial and we’ll have to rethink this and re-do it. Details in post.
Today was Day 1 of the Geoduck strip spawn experiment at Taylor Shellfish Hatchery. This wil be one of my side projects for my MS program because I realized I really wanted some hands-on, hatchery work experience! Lots and lots and lots of details (and photos) in the post.
Sam extracted RNA using the Quick DNA/RNA Microprep Plus Kit (ZymoResearch) (post). I don’t think there is currently enough RNA in the libraries to reach the requiremets for NWGC… details in post.
- Help with getting pooled samples submitted for library prep and sequencing to the NWGC
- Think of what crab-related data that I have can be used for FISH 497 course
Today I spoke with Steven about what the results are for the 2015 C. gigas seed paper. I made a new annotated table for the 69 differentially abundant (though not significantly) proteins. Edits to paper.
Between trying to add some edits and clean things up for the 2015 Oysterseed project paper, I’ve been reading protocols for other RNA extraction kits. Zymo Quick-DNA/RNA Mircoprep Plus Kit, Zymo Directzol RNA MiniPrep… Details in post
Yesterday and today I got some more 2015 DIA paper-related tasks done, including making a table that includes the proteins detected above a log2FC of 3.00 and below -3.00, and one that was annotated…
Today I did the RNeasy Plus Micro Kit protocol (without the vacuum) on 12 samples (taken from the 24 that I did last time). Of the 12, only 1 had detectable RNA. The rest were all “Out of Range: TOO LOW”. Details in post below.
Today I got some more input from Emma regarding the reports to export from Skyline to analyze. I exported a new MS Stats report (the built-in one from Skyline) and also made the bioreplicates in the file accurate (details in post). Additionally, I exported a new report that includes information that should help us get to the point of comparing 23C and 29C proteins, and identifying the proteins expressed in total.
- Identify methods of extracting RNA from low input
- Test out more RNA extraction methods
A little bit of progress has been made with the DIA proteomics paper. I am aiming to have the methods section of the paper solidly done by our meeting this Wednesday at 2 pm.
Today we had our first WSG meeting with the goal of having all of the projects wrapped up by April 17th.
Today I tried out the new plan for extracting RNA. It took quite a long time and none of the 24 samples had detectable RNA. Details in post.
Here’s the general extraction plan after feedback from lab meeting today. I am planning on getting started on extractions next Tuesday (I’ll be done with end-of-quarter work and my online SCUBA class by then!). (Edited this post a lot since my original post this morning)
I forgot to do March goals at the beginning of the month… so here’s what my goals are for the rest of the month! Classes are over this Friday, and I’m not going anywhere for the break, so I think I can get this done!
Today I extracted RNA from tube 516-2 in two different volumes: 10ul and 50ul. The tube only had about 62ul of sample slurry remaining, so I was only able to do 10ul and 50ul. I used the same protocol as what Steven and Shelly did last Friday. I saved the gDNA and eluted it to get the DNA concentration from the samples, and also got the RNA Qubit results. There was RNA in both, and DNA in both. When comparing the amount of RNA extracted to the amount of starting material, there is higher yield when using 10ul of sample than the 50ul of sample. More notes detailed in post.
Today, Steven, Shelly, and I worked together on extracting RNA from bairdi hemolymph pellets using the Qiagen RNeasy Micro Plus Kit with QIAshredder columns to aid in homogenization. Instead of using the whole pelleted sample like I have always done in the past, we let it thaw at room temp, centrifuged at full speed for ~30s, then sampled out 50ul of the hemolymph/RNAlater slurry into new tubes. The reasoning for this is to see if maybe using the whole tube contents resulted in clogging the spin column tubes. Shelly also saved the DNA that got caught on the gDNA spin column, washed, and eluted to quantify DNA using the Qubit DNA Kit. This was to check to see if there was any DNA in the samples. All four had relatively low amounts of DNA, and only one of the four had quanitifiable RNA (rest were TOO LOW (less than 5ng/ul). Next steps are detailed at end of post.
After talking with Steven and Shelly, today we decided I should attempt to extract RNA from supernatant samples using the Trizol LS protocol. This post contains the steps that I performed with pictures of some steps, and results (no RNA in final samples :(…). Overall current plan is to take 4 crabs from Day 26. Use Qiagen RNeasy Kit with QIA shreddercolumn and RNA carrier on the pellets, and use Trizol LS on the corresponding supernatant samples.
This post contains a slightly modified RNeasy Kit protocol. I want to use the Qiagen RNeasy Micro Plus Kit on 4 crab hemolymph pellet samples and the four corresponding supernatant samples (GitHub Issue 577). The goal is to see if there is any RNA in the supernatant samples and if that can help increase the RNA yields for each crab sample. The supernatant samples (~1ml each) will be transfered to 15ml falcon tubes and the volumes of reagents likely need to be changed (GitHub Issue ). I will be using RNA carrier for both supernatant and pellet samples. (Sidenote: just realized I didn’t use QIAshredder columns when I did the RNeasy protocol previously. I have added that to my figure.)
Spoke with Sam the other day about everything we’ve tried in regards to extracting RNA from our crab hemolymph samples. He shared a lot of ideas of potential next steps for figuring out ….
2015 Oysterseed DIA
- continue to work on the paper and figures
- get feedback from people
Today I tried out the Trizol LS Reagent RNA extraction protocol on two geoduck (P. generosa) hemolymph and two oyster (C. virginica) tissue samples. (GitHub Issue #533)
Today I also tried the Qiagen RNeasy Micro Plus Kit on geoduck (P. generosa) hemolymph (2 samples) and oyster (C. viriginica) tissue (2 samples).
Today I tried the TrizolLS Reagent extraction again, but added a vortex (5s) step after the addition of chloroform to the samples. Throughout the extraction, the samples looked pretty similar to how they did during my last attempt, and the Qubit results were the same: “out of range”.
Today I tried out the Trizol LS Reagent extraction on three samples from Day 26. I used 1ul of each sample to run on the Qubit (RNA High Sensitivity), and all three samples were “out of range”. Images and more details in the post below:
Today I’m trying to get started on the new BLAST +2.81 that has new databases and improved performance. This is pretty exciting because once I figure out how this works, I’ll be able to easily get taxonomy information like Order, Class, etc. I’m attempting what I believe is the first step in this process: trying to get the taxid for “Decapoda”. Details below of resources used and what I did.
Today I tried out the Trizol LS Reagent extraction, but the centrifuge in the 4˚C in FTR 213 had an “Err 3” message, which happens when there’s an error in the rotational speed measurement system (GitHub Issue #542). I’ll put a different centrifuge in the 4C and re-try the protocol on Wednesday. This post includes my protocol for the Trizol LS Reagent extraction.
Today I went through my R script for the RobertsLab/project-pacific.oyster-larvae project. I have a Volcano Plot and a Comparison Plot. I also did some data process plots: Profile Plots and QC Plots. I am working on updating the MS Stats protocol, and will include all the resources I used to create those plots. Additionally, I am beginning to read through the Oyster-Larval-Proteomics-2015 paper and am working on finding some new relevant citations. My goal for the month is to finish this project and paper!
2015 Oysterseed DIA
- Finish data analysis
- Edit paper with new methods, etc.
Today I checked on my finished BLAST on Mox with my assembled C. bairdi transcriptome and the
nt taxonomy database. It finished after three-ish days, and the file was large. However, all of the taxonomy cells were “N/A”. I made a GitHub Issue, and got some input and am now BLAST-ing again. Additionally, Sam posted a link for me to look into to get Order, Family, etc. taxonomy information on my next BLAST that I will get going before I leave for CA.
Today I ran the bioanalyzer on the batch of 8 samples from Day 26 (cold, infected) from Nov 21st. Results don’t look super great, and the ladder didn’t show up.
Today I am continuing my work in MS stats and working with the exported Skyline report. I updated the protocol to document what I have been doing to get where I am. I am in contact with Meena from MS stats, who is helping me with an error that keeps coming up in the R code. Once that is fixed, I’ll be able to analyze the data and make some figures/tables.
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:
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.
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.
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….
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.
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!
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.
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.
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.
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
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.
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.
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!
My Trinity job failed and it turns out it’s because I was missing a
/gscratch/srlab/programs/Trinity-v2.8.3/Trinity. I fixed that and then re-sent the job to Mox. The new script is called 20181101_Cbairdi_trinity.sh and it lives in my
/gscratch/srlab/graceac9/jobs/ directory on Mox.
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
- Create new R scripts for adding new Qubit results data to a master file combining hemolymph sampling data and RNA extraction results data
- Publish new Crab Meeting episode
- Record new episode with library plan
- Record an interview with Maddie Shipley (find a time to meet)
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.
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.
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.
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.
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.
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.
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.
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.
Today I extracted RNA from four practice samples using Tri-reagent, and without lyophilizing (lyophilizer is out of commission again for now).
- 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)
Today I submitted DecaPod RSS feed to Apple for approval to be published on iTunes! I also picked out some tubes for Tri-reagent RNA extraction without lyophilizing (it’s broken again) for Monday or Tuesday next week to get things moving again.
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.
Today I started using the lyophilizer on the samples I picked out (pellet and supernatant) and the pooled sample of the ones from Sam’s Qiagen RNeasy extraction that had “Out of range” Qubit results.
Today I edited and published DecaPod S1E11: Crab Meeting #5. I also have a plan for the format of the podcast.
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.
2015 Oysterseed Project:
- Figure out Skyline DIA with Emma (plan for 9/6/18)
- Analyze 2015 Oysterseed data
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.
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.
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. :)
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.
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.
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.
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.
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.
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.
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.
- 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
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.
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).
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…
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.
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.
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.
This post is a summation of what I did this week. I made the 3 pools for RNASeq (speed vac will happen Tuesday when both Sam and I are in), and am starting to create an ultimate master crab spreadsheet with ALL the data that we have on this project.
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.
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.
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.
Today I went through all my setting and files in Skyline for the 2015Oysterseed proteomic project and re-did my Skyline Daily peak-picking error rate determination. I still got an error rate nearing 50%, so per Emma’s suggestion, I submitted the issue to Skyline’s support page.
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.
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.
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.
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.