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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Laura’s Notebook: February 2019 Goals

Accomplishments last month:

• Submitted geoduck proteomics data to PRIDE
• Published skyline project files on panoramapublic
• Submitted geoduck eelgrass revision
• Received feedback on polydora paper from Teri King
• Oly 2017 paper:
  • Began draft introduction
  • Finished draft discussion, methods and results …
    • Received feedack from Steven, re-assessed data to include, changed paper’s angle, plots
• Submitted travel grants for NSA conference
• Agreed to WA Sea Grant symposium, but it was postponed due to gov’t shutdown. Will likely participate next fall.

Things to do, this short month

• Oly 2017 paper:
  • Final edit of methods, results rewrite
  • Finish disucssion rewrite
  • Finalize plots
  • Finish Introduction
  • Draft abstract
• NSA presentation, on Oly 2017 OA / QuantSeq project
  • Need full 12 minute presentation
  • Need 3.5 minute, abbreviated proposal, maximum of 5 slides for student spotlight session
• Bipass proposal … getting antsy
  • Incorporate final geoduck paper
  • Draft list of proposal options with details
  • schedule deadline

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Laura’s Notebook: January 2019 Goals

• Submit geoduck proteomics data to PRIDE
• Publish skyline project files on panoramapublic
• Submit geoduck eelgrass revision
• Format polydora paper for JSR
• Submit polydora paper to JSR
• 2017 Oly paper, “Transgenerational carryover effects of parental low pH exposure in the Olympia oyster (Ostrea lurida)”
  • Identify journal
  • Finish Discussion
  • Finish Introduction
  • Write abstract
• Bipass proposal
  • incorporate final geoduck paper, olympia transgenerational paper
  • draft list of proposal options with details
  • schedule deadline
• Email Deborah about WA Sea Grant event
• Poster for Sea Grant – update geoduck poster, OR do Oly QuantSeq project

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Laura’s Notebook: Assessing Oly gonad transcriptome assembly

The Trinity assembly is complete. Today I inspected it using transrate, in addition to running blast over the weekend to annotate genes using the Uniprot/Swissprot database. Transcriptome assembly quality, as per transrate using the trimmed/normalized reads, seem sub-par, as percent good mapping is only 10% … but I’ll investigate further. Check out my Jupyter notebook for more details: transcriptome-assess-annotate.ipynb

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Sam’s Notebook: New Notebook

0000-0002-2747-368X

I’ve migrated my notebook to here:

• https://robertslab.github.io/sams-notebook/

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Kaitlyn’s notebook: Enrichment and Protein abundance heatmaps

These heatmaps were created by using the BP-FAT file created by DAVID when I entered my uniquely clustered proteins with a background of all detected proteins (in silos 3 and 9).

• Enriched processes merged back to a protein based on Uniprot Accession IDs
• Protein abundances are values in heatmap
• 

These heatmaps show how the protein abundances change over time for proteins whose Uniprot accession codes are associated with enriched BPs represented by parent terms which were given by DAVID during enrichment analysis.

I also included the heatmaps clustered by time below the first pair of plots.

The patterns of abundance seem to be very similar between the two silos, but silo 3 tends to have higher abundances than silo 9 except with platelet degranulation.

---

 

The days are clustered in the plots below. If we look only until the second node, we see  three main groups for Silo 3:

1. Day 13
2. Days 7, 5, 11, 9 
3. Days 15, 0, 3

and two main groups for Silo 9:

1. Days 0, 3, 9   
2. Days 15, 13, 11, 7, 5

Based on this, we can see that protein abundance patterns are different between the silos, but we knew this already since these proteins were selected based on differential clustering before. The new information we can see is how the processes the proteins are linked to shift based on time.

This was done with the BP-FAT file because it had the fewest enriched processes and would be the easiest to work with and view as a test attempt. Here is the code I made.

Laura’s Notebook: Denovo Oly gonad transcriptome assembly (attempt 1)

Today I got comfortable using the Mox (Hyak) supercomputer, created my directories, and queued a transcriptome assembly using Trinity.

For details, please see my notebook: trinity-setup-on-mox.md

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