Looking back at Tophat

RNA-Seq: Tophat via iPlant
http://onsnetwork.org/halfshell/2015/01/08/rna-seq-tophat-via-iplant/

GTF available at
http://eagle.fish.washington.edu/trilobite/Crassostrea_gigas_ensembl_tracks/Crassostrea_gigas.GCA_000297895.1.24.gtf

Though likely an updated version @ http://metazoa.ensembl.org/Crassostrea_gigas/Info/Index

Laura’s Notebook: July 18 2018, O. angasi conditioning

Question: does temp/food conditioning method used on O. lurida work on Ostrea angasi?

No standard operation procedure is in place to get O. angasi to reproduce in the hatchery. This makes for difficult production. There is interest to see whether a highly controlled temperature/feeding regime can successfully increase their gonad condition, which could allow for induced, synchronized spawning. If I’m lucky some of them will spawn during the conditioning phase.

Oysters arrive

On June 20th adult oysters (age ~3-5 years) from Port Stephens and Merimbula (northern and southern extent of New South Wales) arrived into the hatchery. They were acclimated to hatchery conditions in the same tank for 5 days. 25 oysters from each group were peeled off for the heat shock experiment, 20 of which were sampled for time=0 gonad condition (sampling #1).

Experimental design

Three temperatures are being tested: 18C (control), 21C, and 24C, 4 tanks per temperature, 2 per oyster source. In each tank is 12 oysters, and each tank has a separate programmable heater. Beginning June 26 temperatures were slowly raised to the treatment temps. The tanks are 200L with static filtered seawater (filtered to 1um), aerated, and water changed every other day. Oysters are suspended in mesh bags on the side of the tanks and fed ad libitum a ~50%-50% mix of diatom (C. muelleri) and flagellate (Tiso or Pav) – food rations are being recorded, and has been ~10-20L per tank per day, from algae that is 1-2M cells/mL.

Daily maintenance

On water change days oysters are removed from tank, rinsed with fresh water, checked for mortality, and held for ~1-2 hours out of tank during cleaning. This exposure is deliberate, as I have noticed that O. lurida frequently spawned after cleanings. I am using 2 tanks per oyster bag, so that I can fill new tanks 1-2 days prior to the water change, allow the water to warm to room temperature, and maintain a consistent temperature for the oysters. The old water is drained over a 80um-100um screen to check for released larvae or eggs.

Sampling 2: conditioning for 2 weeks at temp

On July 16th, 3 weeks after beginning the conditioning trial and 2 full weeks minimum of treatment temp, I sampled half the oysters from all tanks (n=6 per tank, n=12 per source, n=24 per temperature). Whole weight after shucking/draining, and shell weight were collected to estimate condition index. Tissues sampled included:

  • -80 & RNAlater: gill, mantle, gonad
  • fixed for histology: gonad
  • -80 only: gut/gonad complex

Oysters were also imaged prior to sampling and checked for brooded larvae/embroys. 1 oyster was brooding embryos – Merimbula 24C (tank 1).

Sampling #3 will occur on July 30th – 2 weeks after sampling #2 and 4 full weeks at temp.

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Grace’s Notebook: Speed Vac Pt. 1 + Testing Advancing Peak Picking in Skyline

CRAB RNA POOLS: Today I started using the Speed Vac on the three pools with Sam. They were running on low temperature from 10:30am to 1:15pm. Not much liquid had evaporated. From 1:15pm-3:15pm they were run on medium temperature. Still not enough liquid has evaporated, so Sam will put them in the Speed Vac when he gets to FTR tomorrow morning. SKYLINE: I tested out the tutorial that the people from Skyline suggested I try out. Not sure how it turned out honestly, but Emma said she can take a look at it with me tomorrow.

Speed Vac

Started at 10:30am on low temperature (room temperature)

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Checked at 1:15pm. Still too much volume. Increased temp to medium

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Checked on it at 3:15pm. Still too much volume

Sam offered to put it back in the Speed Vac at medium temperature tomorrow morning when he gets in to FTR. It will hopefully be done by lab meeting. Because there is some precipate in the tubes (precipitate could be salts from the RNA isolation process or un-dissolved RNA), we will mix and Qubit the pooled tubes to get an accurate RNA concentration reading and then adjust the volume for the pools if need be. The CORE facility needs “RNA normalized to a minimum of 20ng/uL with a total volume of 50uL.” (GitHub Issue #184)

2015 Oysterseed Project: Skyline trouble-shooting

My error rates from Step 5b of the DIA Protocol were around or above 50% every time I tried it. Emma prompted me to contact the Skyline Support with my issue.

I wrote on their support page and they responded that I might try their Advanced Peak Picking in Skyline. I tried out their tutorial today with their example files. They don’t have very detailed instructions in the DIA section.

Emma offered to go through it with me either tomorrow after Sam’s birthday lunch or next week when I come back from my CA trip.

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