Grace’s Notebook: Bairdi hemolymph extraction plan (work in progress)

Here’s the general extraction plan. There will be a final version of this extraction plan of actually selecting the tube numbers, and taking into account whatever feedback I get.

Number of samples to process

Based on previous RNeasy processes (Feb 15th, and Feb 20th), we have decided that using 50ul or less of the hemo slurry is best with the RNeasy Kit.

I’ll use 20ul of the hemolymph slurry from about 150 individual sample tubes. (Number of tubes processed will depend on the RNA yield we get)

I’ll process 8 samples at a time, and will select tube numbers such that each infection status and temperature treatment group has 8 samples selected, along with 4 back-up tubes in case there isn’t detectable RNA in any of the samples.

To start, we’ll do 12 sets of 8 tubes, running 1ul of each sample on the Qubit at the end of each set, keeping track of which pools need extra RNA. Then, after the 12 sets of 8, I’ll collect whatever extra tubes we need from the back up list that I’ll make and decide how many additional sets need to be processed.

The goal is to get to the point where I have 12 pooled samples, each with at least 1000ng RNA in 50ul of DEPC-treated H20.

Protocol preparation

Q: Should I prep huge containers of reagents or should I prep them fresh before each extraction?

70% ethanol
80% ethanol
Buffer RLT plus 2-BME


  1. Add 350ul of Buffer RLT Plus + B-ME solution to both samples
  2. Vortex to mix
  3. Transfer lysate to QIA Shredder column with 2ml collection tube. Centrifuge 2min at full speed
  4. Transfer flow-through to gDNA Elimninator column with 2ml collection tube. Centrifuge for 30s at 12,00 g. Discard column. Save flow-through. Also save gDNA column for later use.
  5. I measured the amount of flow-through for both samples and added that same volume’s worth of 70% ethanol. THe 10ul starting material sample had 340ul, so I added 340ul of 70% ehtanol. The 50ul sample had 345ul, so I added 345ul of 70% ehtanol. Mix by pipetting.
  6. Transfer sample (including any precipitate that may have formed) to RNeasy MinElute column. Close Lid. Centrifuge 20s at 12,000g. Discard flow-through
  7. Add 700ul Buffer RW1 to RNeasy column. Close lid. centrifuge 20s at 12,000g. Discard flow-through.
  8. 500ul Buffer RPE to RNeasy column. Close lid. Centrifuge 20s at 12,000g. Discard flow-through.
  9. Add 500ul 80% ethanol to RNeasy column. Close lid. Centrifuge 2min at 12,000g. Dsicard collection tube and flow-through.
  10. Put RNeasy column in new 2ml collection tube. Cut off RNeasy column lid. Keep tube open and centrifuge at full speed for 5min. Discard flow-through.
  11. Put RNeasy column in new 1.5 ml collection tube. Add 14ul RNase-free water (from red-capped aliquotted tube) to center of membrane (I missed the center for the 50ul sample). I forgot to close lid, so the tubes were centrifuged open for 1min at full speed.

Run 1ul of each sample tube on the Qubit using RNA High Sensitivity

Upload the Qubit results from each set of 8 immediately to my laptop, add the tube numbers manually, and save.

from Grace’s Lab Notebook

Grace’s Notebook: 2nd Half of March goals

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!

Crab Project

We have identified a good method of extracting RNA from the hemolymph slurry (if there is hemolymph in the sample).

  1. Order more RNeasy Kit and QIA shredder columns for extractions
  2. Create an extraction plan (use 20-25 ul of slurry)
  3. Once kits arrive (or an arrival date is estimated) put on lab calendar the lab days (so whoever is around to help can help if they want)
  4. Extract RNA to create all the pools (12 pools)
  5. Send pooled samples (50ul samples with at least 20ng/ul (1000ng) of RNA) to NWGC for library prep and sequencing

2015 Oysterseed DIA

  • Create Venn diagram comparing the proteins expressed between the two treatments
  • Other basic comparisons between the two temperatures

from Grace’s Lab Notebook

Yaamini’s Notebook: DML Analysis Part 27

Resolving DML and DMR visualization issues

TL;DR Certain things are a dumpster fire so it’s time to put out the dumpster fire.

Quick overview

I looked at two different chromosomes to see what was happening. Here are some things I learned:

  1. The gene background is hot flaming garbage.



It should just be CG motifs that have 3x coverage or more, but instead it includes multiple loci that aren’t cytosines.

  1. The DML overlap CG motifs better.



I mildly trust the DML track.

  1. The 100 bp DMR are inconsistent



Sometimes DMR include mulitple CG motifs or DML, but sometimes they don’t. I think using a step size of 100 bp may be influencing this (more on that below).

  1. The 1000 bp DMR make no sense



The 1000 bp DMR are heavily influenced by regions where one sample is hypermethylated in that region, but other samples aren’t methylated there (or have no data). I did find an instance or two where multiple samples were hypermethylated in a 1000 bp DMR, but these were rare over the two chromosomes I looked at.

Generate 5x and 10x DML tracks

At this point, I mildly trust DML. We’re currently using 3x coverage for analyses, but previous papers have used 5x coverage. We decided to look at 5x and 10x DML tracks as well.

In this R Markdown file, I created 5x and 10x DML bedfiles (found here). Steven pointed out that he used destrand = TRUE in his unite command, and I did not. My current stranded output includes + or – indications for forward and reverse strands, but normal discussion of methylated loci does not include strandeness. According to the methylKit manual, destrand = TRUE provides better coverage for CpG methylation. I created 5x and 10x coverage tracks using both destrand = FALSE, the default, and destrand = TRUE. To visualize everything in my IGV session, I also created 5x sample coverage tracks, found here.

  1. Some DML were retained through all coverage types.


I didn’t see much loss going from 3x to 5x DML tracks, but there were more DML lost from 5x to 10x.

  1. Some DML were ony present in destranded 5x or 10x tracks.


This is probably a result of the increased coverage in the destranded tracks.

  1. I also found something really weird.


This CpG had a DML on the forward strand in the 3x, 5x, and destranded tracks, but on the reverse strand in the 10x track. Not sure how this can happen…

Going forward

  1. Figure out which DML track to use for remaining analyses and characterize DML locations in that track
  2. Describe methylation irrespective of treatment
  3. Work through gene-level analysis
  4. Figure out what’s going on with DMR
  5. Figure out what’s going on with the gene background

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from the responsible grad student

Laura’s Notebook: March goals

Laying down my monthly goals midway through March and towards the end of the quarter means that I need to be realistic/conservative.

Accomplished February/early March:

  • Re-worked Oly parental OA exposure paper methods/results/discussion
  • Sent Polydora paper to co-authors for final comments
  • TA duties maintained
  • Secured TA-ship for next quarter (shellfish biology with Jackie)
  • Prepared presentations on Oly OA parental effects
  • Presented twice at Aquaculture 2019 in New Orleans, the triennial NSA meeting. Presented Oly parental exposure & QuantSeq data in the Mollusc restoration session, and also in the Student Spotlight competition.
  • Fulfilled my Student Recruit Co-chair duties at the NSA meeting!
  • Identified how to use remaining grant money – will broaden my larval QuantSeq data, and perhaps do some samples from the 2018 winter temp/food experiment to look at gene expression in high/low performing larval families.
  • Also decided to keep an eye open for opportunities to sequence adult somatic tissues after Oly OA exposure.

To do, rest of March:

  • Outline of bypass proposal – start drafting during spring break downtime
  • Refine discussion section of Oly 2017 paper, outline introduction
  • NSA quarterly newsletter for Leroy
  • Start testing QuantSeq analysis pipeline with Oly genome, Salmon (removing the multiple read/transcript auto-correct), Trinity’s isogroup designation file.
  • Plan QuantSeq sample selection, RNA isolation, library prep, sequencing, etc.
  • Help/facilitate Alanna with RNA extractions
  • Clean up teaching lab
  • Revise/finalize cover letter for Polydora paper

from The Shell Game