Laura’s Notebook: First day of tissue prep, Alanna captstone / acute OA shock trial

Today Alanna and I got started processing juvenile Olympia oyster whole body tissues for RNA extraction. We are using the South Sound offspring (from Oyster Bay Cohort 1), from the 6C parents that were exposed to 7.3 pH and those that were unexposed, and which were held in control pH tanks (8.0), and acute low pH (7.0) for about 6 hrs.

We are processing 8 samples at a time, 2 per each treatment. First step is tissue disruption and homogenization in liquid nitrogen using a mortar and pestle, which we did today on the following samples:

Sample # Population Parental Treatment Offspring Treatment
100 SN 6-Amb control
106 SN 6-Amb control
112 SN 6-Amb acute low pH
115 SN 6-Amb acute low pH
124 SN 6-Low control
126 SN 6-Low control
136 SN 6-Low acute low pH
136 SN 6-Low acute low pH

Note: after grinding in liquid nitrogen, no more than 5 mg were allocated into tubes, then 350 uL of Buffer RTE Plus was added, the mixture was vortexted vigorously, then all was transferred to the QIAshredder columns, and spun at maximum speed (21,300 rcf) for 2 minutes. The homogenate, which the protocol says can be held in -70 for “months”, was then put into the -80 for RNA extraction tomorrow. samples are held in collection tubes in the -80, rack #1, column 2, row 3. remaining dry ice is in the middle -80 freezer, top shelf, in a styrofoam cooler.

The oysters are small, about 5mg total tissue. I was able to save about 1/2 the ground tissue for only some of the samples (106, 112, 115, 126). If we get enought RNA with less than 5 mg of tissue, I will see if Alanna can do this for the rest of the samples.

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Grace’s Notebook: RNeasy Kit Tube 516-2 starting sample testing

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.

Prep before extraction

Made 70% ethanol
Made 80% ethanol
Made Buffer RLT Plus and B-ME solution (originally for four samples- I thought I was going to have more 516-2 slurry left over)
2 mL Buffer RLT Plus
20ul B-ME

Protocol

  1. Added 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.

DNA elution
img

Results

RNA
Google sheet

10ul starting sample volume –> eluted 14ul RNase-free water –> ran 1ul on Qubit RNA HS –> 4.6ng/ul
50ul starting sample volume –> eluted 14ul RNase-free water –> ran 1ul on Qubit RNA HS –> 14 ng/ul

When comparing the starting material volume to the RNA yield, you get more RNA from the 10ul starting sample volume.

Shelly’s napkin notes:
img

DNA
Google sheet
10ul starting sample volume –> eluted in 100ul Buffer AE –> ran 1ul on Qubit BR DNA –> 2.28ng/ul
50ul starting sample volume –> eluted in 100ul Buffer AE –> ran 1ul on Qubit BR DNA –> 12.9ng/ul

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Shelly’s Notebook: Fri. Feb 15, 2019; Diploid and triploid global DNA methylation and crab RNA

Diploid and triploid global DNA methylation

I re-did the global DNA methylation triploid/diploid pilot analysis in R to compare the results to the data Ronit and I generated Wednesday Feb 6.

The pilot data showed diploid mantle has more methylation than triploid mantle. And there is a more dramatic decrease in global 5-mC methylation in response to heatstress in triploid than in diploid mantle.

unnamed-chunk-13-1.png

However, Ronit’s experiment with Ctenidia showed a different trend. Global methylation was not significantly different between diploids, triploids, or triploids exposed to heatstress. But diploids exposed to heatstress showed a significant increase in methylation.

Dip_vs_Trip_HS_globDNAMeth_boxplots.jpg

Since the pilot results are only from two biological replicates in each group, it’s not clear if these trends would persist if more mantle samples were processed.

The pilot data also showed a difference between tissue types, but again it’s not clear if these trends would still be seen if more samples were processed. unnamed-chunk-12-1.png

RNA from tanner crab hemolymph

Went through RNeasy kit with Grace, Steven, Laura, and Alana. Grace had 4 crab samples and Alana was preparing 4 juvenile oyster samples side-by-side.

After the gDNA eliminator column step, I took the gDNA eliminator column and eluted the gDNA off the column following the procedure below adapted from this researchgate post Screen%20Shot%202019-02-15%20at%2011.51.00%20AM.png I ended up spinning at 4C since the room temp centrifuge was being used for the RNA extractions, and I ended up using 50-55C prewarmed AE since that’s was the heatblock was set at.

The DNA and RNA qubit results were as follows: IMG_20190215_152715%7E2.jpg

I saved the DNA samples in my DNA box in the -20C in rm 213.

We still aren’t sure whether the RNeasy columns are being overloaded or if the RNA in the samples is degraded. So next Grace is trying to isolate more RNA from sample 516 and will be testing 10uL vs. 50uL vs. 100uL of pellet slurry (starting material). This should hopefully determine the appropriate amount of starting material to use.

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Grace’s Notebook: RNeasy Kit with hemo pellets using QIAshredder columns

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.

Prep before protocol

Samples

We used the corresponding pellets from my supernatant extractions:
507-2
510-2
513-2
516-2
img

These tubes are the second out of three.

Steven let them thaw to room T, centrifuged at full speed for ~30s, then pipetted out 50ul of each sample into a new labeled, RNAse free tube (from RNA station by Sam’s desk). I put the original tubes containing the rest of the samples back to their spot in the -80.

Solutions:

I made a solution of B-ME + Buffer RLT Plus for my four samples in a falcon tube from the RNA station.
2mL Buffer RLT Plus
20ul B-ME

I made 70% ethanol using a serological pipet and the ethanol and 0.1% DEPC-treated H20 in a falcon tube, all from the RNA station.
7mL ethanol
3mL DEPC-H20

NOTE: I should have also made 80% ethanol. I had to go take a midterm, so Steven and Shelly carried on the protocol after step 3 of protocol.

I also put 1ml of RNase-free H20 in the Carrier RNA and let it sit at room T for a bit. We decided not to use it, so I put it in the -20C in 209 (labeled lid with date and my initials). This can now act as a stock solution to make dilutions from for future use.

Protocol

  1. Added 350u of the B-ME Buffer RLT plus solution to each tube
  2. Vortex
  3. Homogenize by putting lysate into QIAshredder columns. Centrifuge for _____ at full speed.
  4. Transfer homogenized lysate (in collectin tube) to gDNA spin column. Centrifuge for __ at full speed. Keep column to check to see if there’s DNA (more on this later), and KEEP THE FLOW THROUGH.
  5. Add 350ul of 70% ethanol to flow-through. Pipet to mix.
  6. Transfer sample to RNeasy MinElute column. Close lid. Centrifuge for __ at full speed. Discard flow-through.
  7. Add 700ul of Buffer RW1 to RNeasy MinElute spin column. Close lid. Centrifuge for __ at full speed. Discard flow-through.
  8. Add 500ul Buffer RPE to RNeasy MinElute spin column. Close lid. Centrifuge for ___ at full speed. Discard flow-through.
  9. Add 500ul of 80% ehtnaol to RNesay MinElut spin column. Close lid. Centrifuge for __ at full spind. Discard collection tube and flow-through.
  10. Place RNeasy MinElute column in new 2ml collection tube. Open lid of spin column and centrifuge for ___ at full speed. Discard collection tube with flow-through.
  11. Place RNeasy MinElut column in a new 1.5ml collection tube. Add 14ul RNase-free water (aliquoted tubes from -20) directoly to center of the column membrane. Close lid. Centrifuge for ___ at full speed to elute RNA.

Then run 1ul of sample on Qubit using the Qubit RNA High Sensitivity reagents.

gDNA spin column

Shelly’s comment on my GitHub Issue describes how to save the DNA from the gDNA spin column. I’ll have to ask to see if she followed these ratios. She eluted DNA and then ran the DNA on the Qubit using the DNA reagents.

Results:

img

I placed the RNA tubes in the -80 in my protocol testing box (rack 5, column 4, row 3).

Notes

Qubit was done using 1ul of sample.

While this protocol was being performed, Laura and Alanna were both working on oyster tissue using the same protocol with 5mg of ground up oyster body. Their results are below mine. With the same protocol, they got RNA that was above the maximum detection limit for the Qubit RNA HS kit, and also got high DNA from their sample.

The crab hemolymph had relatively low DNA in all four samples, and only had RNA (7ng/ul) in sample 516-2.

There could be RNA in 507, 510, 513, but it might be too low to detect (less than 5ng/ul) and those samples may have required more than 50ul of the pellet in order to extract RNA.

Next steps

After I got back from my midterm, I saw the results of what happened while I was gone. Steven, Shelly and I talked and decided the next logical step is to run this kit again using more of the pelleted hemolymph from tube 516-2 (because we know that there is RNA in there).

I will use 4 volumes of that pellet:
10 ul
50 ul
100 ul
150 ul

If there isn’t enough to do all four, then I’ll just do 10, 50, 100 ul.

I will thaw the sample, but I’m not sure if I should centrifuge it, too? Maybe I could just vortex?

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Grace’s Notebook: Supernatant Trizol LS RNA Extraction attempt

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.

Here’s what Steven, Shelly and I identified as the current plan:

img

Use 4 crabs from Day 26 – making sure that the crabs chosen have 2/3 sample tubes left (some crabs from day 26 only have one tube left!!)

Use Trizol LS to attempt to extract RNA from the supernatant from those 4 crabs. (ATTEMPTED THIS TODAY- DETAILS BELOW)

Use Qiagen RNeasy Kit with QIAshredder columns for homogenization, and use RNA carrier on the pelletted samples from those 4 crabs.

If there is RNA in supernatant, we could combine pellet and supernatant to get higher yields of RNA per sample… (GitHub Issue with Crab Project Next Steps).

Preparation before protocol:

Selected 4 samples from Day 26 that still had 2/3 samples left per crab. The samples selected were:
507
510
513
516

Today I only used the supernatant (RNAlater), which looked like this in the -80:
img

I let the samples thaw. Samples were pretty thick and almost gel-like in consistency:
img

Each tube contained ~1.5ml of supernatant. To keep the ratios simpler, I separated the samples into four tubes, so that each sample was now 4 tubes of 250ul of supernatant each (16 tubes total).

Protocol Part 1: Lyse samples and separate the phases (link to manufacturer protocol: here):

  1. Add 750ul of TRIzol LS reagent to each tube
  2. Homogenize by pipetting up and down (5x)
  3. Centrifuge 5mins at 12,000 g at 4C. Transfer clear supernatant to new tube. (This step isn’t necessary, but while I was doing the protocol, I couldn’t remember if we decided to do it or not… so I just did it. Question: maybe I should try protocol again without this step? Maybe I’m losing RNA during the transfer step?)
    After the centrifuging in step 3, all samples looked like this:
    img

So before I went on, I did my best to discard the pink layer and only transfer the clear part to a new tube. Then I continued:

  1. Incubate for 5 mins
  2. Add 200ul of chloroform.
  3. Incubate for 2-3 mins.
  4. Centrifuge samples for 15mins at 12,000 g at 4C. After this, the samples should separate into three phases: a lower red phenol-chloroform, interphase, and an upper colorless aqueous phase.
    Here’s what some samples looked like that I wasn’t able to avoid transfering over the pink in step 3:
    img
    Here’s what some samples looked like that I was able to only transfer the clear in step 3:
    img
  5. Transfer the aquaous phase containing the RNA into a new tube.
    During this step, the volumes that were transfered for each sample weren’t equal. For one of the tubes from sample 516, I accidentally discarded about 200ul of the aqueous phase… but was able to transfer a little less than 200ul afterwards because there was still some left. Question: should I have aimed to transfer the same volume for each tube?

Protocol Part 2: Isolate RNA

Step 1: Precipitate the RNA

  1. Add 500ul of isopropanol
  2. Incubate for 10 mins
  3. Centrifuge for 10 mins at 12,000 g at 4C. Total RNA forms a white gel-like pellet at bottom of the tube.
    I couldn’t see any gel pellets, but I continued the protocol as though there were some:
    img
  4. Discard supernatant.

Some notes on this section:
During this part, I posted an issue on GitHub (#581) asking when I should recombine the 4 tubes into one sample. Sam suggested I do it during the precipitation section by centrifuging 1 out of the 4 tubes, discarding the supernatant, and then adding the contents of the second tube of the four to that first tube, centrifuge, and discard the supernatant, and so on.
Before seeing that Steven suggested I talk to Shelly as well, I started doing what was suggested and centrifuged 1 out of 4 of each of the samples.
While those 4 were in the centrifuge, Shelly and I talked and she made the point that all of the volumes in the remainder of the protocol are based on the initial volume of Trizol LS used (in this case, 750ul per sample). By combining 4 tubes into 1 sample, I’d then have to quadruple all the volumes of the remaining reagents for the rest of the protocol, so instead we decided it would be easier to combine samples during the solubilization step of isolating RNA (will describe later).
As a result of what’s stated above, 12 of the samples incubated at room temperature for 20 mins, and the four samples that I first centrifuged sat at room temp (with superantant still in tubes) for 10mins after centrifuging…

Step 2: Wash the RNA

  1. Resuspend (by pipetting up and down) the pellet in 1ml of 75% ethanol.
  2. Vortex briefly (3 s), then centrifuge for 5mins at 7500g at 4C.
  3. Discard supernatant.
  4. Air dry pellet for 5-10 mins.

Some notes on this section:
I was able to see something at the bottom of all the tubes – a grey-ish white pellet of something:
img

During the drying step, I discarded the supernatant and left the samples to dry for 10 mins. When I looked at them after 10mins, I realized there was still too much liquid, so I used a smaller pipet to pull off more supernatant and then let them air dry for 10 more mins… Not sure if this was a bad idea.

Step 3: Solubilize the RNA

  1. Resuspend the pellet in 20ul of RNase-free water – I used 0.1% DEPC-treated water.
  2. Incubate on a heat block at 55C for 15 mins.

Some notes on this section:
For the resuspension, I did what Shelly and I discussed and used this step to recombine the 4 tubes for each sample into 1. I did this by starting with one of the 4 tubes, adding 20ul of RNase free water, pipetting up and down to resuspend pellet, then sucked it all up into the pipet tip, and deposited it in the second of the four tubes. Then I pipetting up and down to resuspend that second pellet, increased the volume on the pipet tip so that I could transfer the whole sample, and desposited it in the third of the four tubes… and so on. By the end of the resuspensions, each sample tube ended up with somewhere between 24 and 25 ul.

Results: No RNA

I ran 1ul of each sample on the Qubit using RNA High Sensitivity reagents. Unfortunately, none of the samples had any detectable RNA. They all said “Out of Range: TOO LOW”.

I saved the samples anyway, and put them in my RNA extraction protocols test box in the -80 (Rack 5, column 4, row 3).

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Grace’s Notebook: RNeasy Plus Micro Kit Protocol using RNA Carrier and Supernatant

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.)

[code] [sr320@mox2 jobs]$ cat 0212_1230.sh...

[sr320@mox2 jobs]$ cat 0212_1230.sh 
#!/bin/bash
## Job Name
#SBATCH --job-name=geoy-blast
## Allocation Definition 
#SBATCH --account=coenv
#SBATCH --partition=coenv
## Resources
## Nodes (We only get 1, so this is fixed)
#SBATCH --nodes=1   
## Walltime (days-hours:minutes:seconds format)
#SBATCH --time=3-00:00:00
## Memory per node
#SBATCH --mem=100
##turn on e-mail notification
#SBATCH --mail-type=ALL
#SBATCH --mail-user=sr320@uw.edu
## Specify the working directory for this job
#SBATCH --workdir=/gscratch/srlab/sr320/analyses/0212

/gscratch/srlab/programs/ncbi-blast-2.6.0+/bin/blastn \
-task blastn \
-query /gscratch/srlab/sr320/data/geoduck/Pgenerosa_transcriptome_v5.fasta
-db  /gscratch/srlab/sr320/blastdb/Pgenerosa_v071 \
-out /gscratch/srlab/sr320/analyses/1119/geo-tran-v071.tab \
-evalue 1e-20 \
-max_target_seqs 1 \
-outfmt 6 \
-num_threads 28

#sbatch