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.)

Kaitlyn’s notebook: Extracting clusters from heatmap (and a Venn diagram)

Extracting Clusters

I’ve been working on extracting the clusters out from my heatmap and finally succeeded with this R script!

Cluster 1 

Cluster 2

23C

29C 23C 29C
Mean Protein Abundance 181.025 168.728 284.94 301.742

Important note:

  • distfun = function(x) as.dist(1 - cor(t(x), use = "pa"))
    • heatmap3 states that distfun=dist is default but that is incorrect and the above is the appropriate default;
    • gplots does have the correct distfun=dist default so you  must change it if heatmap3 gives a better dendrogram as in this case.

Question: Should I get error rates for the mean protein abundance of each cluster and silo?

I’m currently working on doing the same thing with the ASCA temperature influenced proteins (the ASCA table requires some different reformatting).

Venn Diagram

I had a lot of trouble trying to make a venn diagram in R (here is my attempt…). I did use the previous script to sort out a list of the proteins in each temperature, but ultimately I decided to refer back to Emma’s paper (2) and use Venny (2) the same way she did.

(1) Oliveros, J. C. Venny. An interactive tool for comparing lists with Venn’s diagrams.
https://bioinfogp.cnb.csic.es/tools/venny/index.html (accessed February 13, 2019).

(2) Timmins-Schiffman, E. B., Crandall, G. A., Vadopalas, B., Riffle, M. E., Nunn, B. L., Roberts, S. B. (2017). Integrating Discovery-driven Proteomics and Selected Reaction Monitoring To Develop a Noninvasive Assay for Geoduck Reproductive Maturation. Journal of Proteome Research, 16(9), 3298–3309.doi:10.1021/acs.jproteome.7b00288.

[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

Ronit’s Notebook: Finalized qPCR Plots

I worked on figuring out some of the stats for my qPCR data with Shelly on Thursday. I decided to go with an ANOVA/Tukey’s Honest Significance Difference Test, with the data being normalized using a log transform. Final qPCR plots are attached below–capital letters indicate differences in treatment, asterisks denote differences between individual groups, and capital letters next to ploidy key indicate differences between diploids and triploids. ATP SynthetaseCOX1DNMT1HATHATHaP2HIF1AHSC70HSP90MBD2MeCP2SOD.png

Sam’s Notebook: Samples Submitted – Robertos C.gigas DNA for Whole Genome Bisulfite Sequencing (Genewiz)

Sent Roberto’s 12 designated DNA samples for whole genome bisulfite sequencing (WGBS) to Genewiz. They will do all bisulfite conversion, library prep and sequencing (Illumina HiSeq; 2x150bp; 30x coverage).

Also sent two samples prepared by Shelly (Panopea generosa) and two samples prepared by Yaamini.

Here’s the list of samples:

Roberto

  • 0501
  • 3501
  • 5201
  • 5901
  • 0502
  • 3502
  • 5202
  • 5902
  • 0530
  • 3530
  • 5230
  • 5930

Shelly

  • Tank3-15_16
  • Tank2-025_026

Yaamini

  • YRVA
  • YRVL

Turnaround will probably be around 30 days.

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Kaitlyn’s notebook: oyster seed proteomics comparisons

Comparing ASCA and clustering identified proteins

I modified and reran the clustering code using the technical replicate averages. The code and other plots can be found here. I used the proteins ASCA identified as being affected by temperature.

This is the code I used to compare the analyses. A total of 25 proteins were common between clustering and ASCA analysis. ASCA identified 113 different proteins while clustering identified 8 other proteins. It would be interesting to see how manipulating the dendrogram height cutoff value during clustering affects the latter number (I used a cutoff of 250).

Here is a heatmap of the similar proteins (note the first column is Day 0 followed by 23C days 3-13 and finally 29C days 3-13):

Venn Diagram of 23C and 29C

I’m working on making a better venn diagram in R with just the silo 3 and 9 proteins. Here is a quick and dirty venn digram, but I’m trying to usethe VennDiagram package to improve it.

Note that all 0 values were changed to 0.100 in the technical replicate averages. I removed all values less than 0.600 as these would be undetected in the silo because of the change (there are 6 days of data since were are excluding day 15 because of the temperature malfunction).

This is obviously still a work in progress to create a better venn diagram.

Yaamini’s Notebook: WGBS Samples Part 2

Continuing isopropanol precipitations

While thinking about my WGBS sample preparation last night, I realized that I don’t have enough of my low pH sample! I had a sample concentration of 20.8 ng/µL, but only 14 µL of sample after the Qubit. This means that sample volume was less than 500 ng and unusable for sequencing. After talking to Shelly, I did the following:

Isolate new samples

I needed at least 200 ng of sample to reach the 500 ng minimum. I took 6.3 µL of 9-T2 and 5.5 µL of 10-T3, added them to a new tube, then vortexed the new pooled sample. I put the pooled sample back in the fridge until I needed it.

Reprecipitate supernatant

I took the isopropanol supernatant I saved and added 5.31 µL more sodium acetate and 15 µL of isopropanol. I then spun the tube for 30 minutes at 12000 rpm and 4ºC. When I went to pipet the supernatant off, I couldn’t see a white pellet. I took off the supernatant and disposed of it, leaving a little bit of liquid at the bottom. For an ethanol wash, I added 90 µL of 75% ethanol and spun the tube for 10 minutes at 12000 rpm and 4ºC. Once again, I couldn’t see a clear pellet. I took off as much ethanol as I could and warmed the tube for 2 minutes on a heat block set to 37ºC. I took a little bit of remaining liquid and added it to the samples to the 200 µL of pooled samples I made prior.

With the ethanol wash supernatant, I added 2.5 µL of sodium acetate and 10 µL of 75% ethanol. I then put the tube in the -80ºC for an hour. After the incubation, I spun the tube for 30 minutes at 12000 rpm and 4ºC. Just like the ethanol supernatant, I couldn’t see a clear pellet. I did an ethanol wash with 90 µL of 75% ethanol then spun the tube for 10 minutes at 12000 rpm and 4ºC. After removing the supernatant, I set the tube on the 37ºC heat block for 2 minutes. I used the 200 µL of pooled samples to wash out any DNA stuck to the side of the ethanol wash supernatant tube. I had to run to class, so I left the tube to incubate at room temperature for a few minutes.

Precipitate supernatant and sample combination

Shelly was nice enough to measure my sample concentration! Here’s what she did:

Before I combined the two tubes, one looked particularly cloudy, which is odd. So I combined the samples and precipitated again (40uL sample + 28uL isopropanol + 4 uL 3M NaOAc). Vortexed well. Spun for 30 min @4C. Pipetted off supernatant. There was a white pellet, which probably shouldn’t be so white. I think that indicates a lot of salt. So I did 2x 1mL washes with 75% ethanol each spinning for 30 min. The pellet still seemed pretty white. Then let dry at room temp open cap for 15 min. Then resuspended in 11uL EB (Qiagen, from Sam’s bench), and it was much less cloudy. Then let sit at room temp for 30 min before reading concentration.

The final concentration was 64 ng/µL in 10 µL. I have more than enough sample to send for sequencing!

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Yaamini’s Notebook: WGBS Samples

Preparing C. gigas DNA for WGBS

We’re planning on sending samples for whole genome bisulfite sequencing, and Steven asked me prepare two pooled samples with C. gigas broodstock DNA I extracted. These samples needed to have at least 500 ng of DNA and a concentration of 20 ng/µL. I decided to make 2 pooled samples: one for the low pH group and one for the ambient pH group. For each pooled sample, I used two stage 0 oyster samples (sexually undifferentiated) with the highest yield. This way, I could hopefully use the samples for sequencing again, and I’d have a baseline for DNA methylation without sex effects. Since each pooled sample needed 500 ng, I obtained 250 ng of DNA from each individual sample.

Low pH sample (WGBS-YRVL 2/4)

9-T2: 15.9 ng/µL, 795 ng total in 50 µL. I used 15.8 µL of this sample.
10-T3: 18.3 ng/µL, 915 ng total in 50 µL. I used 13.7 µL of this sample.

The total volume of this pooled sample was 29.5 µL, and the concentration was 17 ng/µL. This was too dilute for sequencing, so Shelly helped me do an isopropanol precipitation to concentrate the sample, modified from her protocol. I added 20.65 µL of isopropanol to the sample (70% of original sample’s volume) and 2.95 µL 3M sodium acetate (10% of original sample’s volume). While I added the reagents to the sample, Shelly placed the centrifuge in the 4ºC fridge. We spun the sample for 30 minutes at 12000 rpm at 4ºC. The DNA was pelleted out and was white because of the salts. I removed the supernatant and saved it in a different tube, just in case (YRVL sup 2/4). I then added 75% ethanol and spun the sample and ethanol for 10 minutes at 12000 rpm and 4ºC. After the ethanol wash, I once again remove and saved the supernatant (YRVL EW 2/4). I kept the sample on a heat block at 37ºC for 2 mintues to dry the sample. While the sample was on the heat block, I also warmed the elution buffer from the E.Z. DNA kit. I then added 15 µL of the elution buffer to my sample and used the Qubit to measure the concentration. My final sample concentration was 20.8 ng/µL.

Ambient pH sample (WGBS-YRVA 2/4)

11-T4: 15.7 ng/µL, 785 ng total in 50 µL. I used 16.0 µL of this sample.
UK-01: 63 ng/µL, 3150 ng total in 50 µL. I used 4.0 µL of this sample.

My final sample concentration was 25 ng/µL in 20 µL. I did not need to modify this sample.

I saved both samples in the fridge so we can send them off for sequencing when UW operations aren’t affected by snow.

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