Water filters stored at -80C in ~1mL of RNAzol RT were provided by Lisa. This is part of an experiment (and Capstone project) to assess RLO viability outside of the host.
The samples were thawed and briefly homogenized (as best I could) with a disposable plastic pestle. The samples were then processed according to the manufacturer’s protocol for total RNA isolation. Samples were resuspended in 25μL of nuclease-free water (Promega).
We need to send half of each sample that we have from Sean Bennett’s Capstone project to Alyssa Braciszewski at UC-Irvine.
This is quite the project! There are ~75 samples, and about half of those are tissues (presumably digestive gland) stored in RNAzol RT. The remainder are RNA that has already been isolated. Additionally, tube labels are not always clear and there are duplicates. All of these factors led to this taking an entire day in order to decipher and process all the samples.
I selected samples from only those that I was confident in their identity.
I aliquoted 25μL of each RNA for shipment to Alyssa.
Tissue samples were thawed and tissue was cut in half using razor blades.
Planning to send samples on Monday.
Lisa has already assembled a master spreadsheet to try to keep track of all the samples and what they are (Google Sheet): Pinto Transcriptome
Lisa provided me with a set of water filters stored in 1mL RNAzol RT @ -80C to isolate RNA from. This is an attempt to assess withering syndrome viability from within the water.
The samples were thawed and briefly homogenized (as best I could) with a disposable plastic pestle. The samples were then processed according to the manufacturer’s protocol for total RNA isolation. Samples were resuspended in 20μL of 0.1%-DEPC H2O.
Samples were stored @ -80C. Will DNase next week.
The sample names are as follows (the ‘C’ is short for “Control”, per Lisa):
Today we sampled the geoduck (Panopea generosa) for genome sequencing. Here is how things went down.
It was an early morning for the clam, peaking out to see a glorious sunrise on the porch.
From there it was off to the lab.
After cleaning the surfaces, Brent sampled tissue.
We started out started out targetting the foot and adductor muscles. These tissues were steriley removed and then rinsed in 1% bleach, followed by Nanopure water. This tissue will be used for genome sequencing as we predict least amount of associated taxa.
Remaining tissues were taken, primarily for RNA-seq and divided into two boxes.
Tubes were labeled on cap with tissue type.
Here is what Box 1 looks like.
Box 2 looks the same however it does not have a heart or style sample.
The only surpise was in sampling, labial palps were identified after we had already sampled a pair.
Despite the indication in this notebook, the samples had not been previously homogenized in RNAzol RT. I thawed the samples, homogenized them and followed the RNAzol RT protocol for total RNA isolation. Here’s the list of samples:
42215 NM1 1
42215 NM1 2
42215 NM1 3
42215 NM1 4
42215 NM1 5
42215 NM1 6
42215 NM1 7
42215 NM1 8
RNA was resuspended in 50μL of 0.1%DEPC-H2O and stored @ -80C in the original box they came from.
Excluding the no template controls (NTC), all samples produced amplification. Will require DNasing before making cDNA.
Related to the qPCR I ran earlier today with these same primers, the efficiencies of the reactions on this plate are significantly better (i.e. normal; >80% efficiencies) than the earlier qPCR. The improved efficiency would also explain why the positive control comes up two cycles earlier on this run.
In the amplification plots below, the positive control reps are the two lines coming up at cycle ~20.