Katie Notebook: Results Organized by Population

7/22/17

Now that I officially have all the oysters sampled accounted for, staged, and recorded I split up my results by population as suggested by Laura. I went through and reviewed all my oysters I had previously labeled as HPM or HPF and, based on the literature, (Oates) decided if they were developing hermaphroditic oysters or or actually oysters going towards being male or female (sperm or eggs were degrading). That gave me only three categories to work with and much more visible results. She was right! There do seem to be more differences between populations than between treatments overall. It also seems like overwintering temperature has a bigger impact than OA treatment does, but that is just at first glance. I’m not sure how to pull any significant results from my figures at this point….my statistic skills need some brushing up on.

Check out these results and see if anything should potentially be combined in different ways. The full updated excel document is posted under Histology staging results.

I first made pivot tables to get all the total distributions of sex within each population and treatment:

Then I organized it all in a new spreadsheet so that figures can be easily generated in the future using any combinations of these results:

Then here are some figures I made that show the comparison between overwintering temperature results! Hood Canal oysters show a significant different between the two temperature treatments, South Sound oysters are pretty similar within the population across temperature treatments, and then the Fidalgo Bay oysters are interesting in that they have the exact same results, but there are no females…

I also started play with OA comparison figures. I could do it in two ways: Compare, for example, both the treatments that were overwintered at 10 degrees but had different OA treatments OR I could compare oysters that had the same OA treatment but were overwintered at different temperatures.

For now I am going to work on making a google doc to begin typing up my methods.

Laura’s Notebook: Second SRM sample prep

Readied last 25 peptide samples for mass spec

0) Labeled mini centrifuge tubes with second batch of samples, and reviewed calculated volumes of sample & Final Solvent to add to each:

1) Borrowed one last 20ul aliquot of PRTC stock from Emma/Jenn – PRO TIP: order mass spec supples way in advance! 2) Added 30ul Final Solvent, vortexted, spun down 3) Pipetted 1.88ul diluted PRTC to centrifuge tubes 4) Pipetted designated volume of Final Solvent, as per my calculates 5) Got samples out of freezer, pipetted designated vol. into centrifuge tubes as per my calculations; vortexed, centrifuged lightly. 6) Transferred mass spec-ready mixtures to autosampler vials; instead of pipetting 15ul I used 14.7, since using 15ul consistently resulted in air bubbles. Despite this, I had to re-pipette samples 54 & 55. 7) Added these newly prepared samples to the sequence table, continuing the pattern of QC, blank, 5 samples, repeat. 8) Loaded the samples on to Tray 2 in the following order:

1	2	3	4	5	6	7	8
A
B	G041	G066	G105	G032	G129	G054	G081	G003
C	G074	G014	G049	G053	G104	G055	G042	G064
D	G073	G057	G007	G070	G001	G071-B	G062
E	GBlank	OBlank
F

9) Added 65ul Final Solvent to the blank vial; there should be ~130ul blank as of 5:30pm today.

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Laura’s Notebook: Checking in on my oysters

Stopped by Manchester for the AM to check on things.

• Downwellers inside were very dirty, holding lots of water but not overflowing. I cleaned them with fresh water.
• I have 9 larval groups left (down from 16). Changed banjos. Water flow and food all looked good, no overflows.
• Water change on SN growth experiment. This week I don’t have time to check these oysters out, but next week I plan to look at them under the scope, and assess whether or not I can count live/dead easily, and how I can keep them separate to potentially extend the experiment (is it even possible?)
• Outside tank:
  • Drained tank
  • Rinsed downwellers and upwellers with fresh water. Downwellers were clean (since Olivia cleaned them on Monday), but upwellers were very dirty. After rinsing them, I uncovered some beautiful oysters! Here is the NF6 Ambient silo, which has a ton of juveniles:
• I received the 1”->1/2” reducing couplers in the mail, so I set up one more downwelling string, and moved the HL-6 Ambient & HL-6 Low 180um setting silos outside.

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Laura’s Notebook: Predicting Retention Times to asses Assay Success

Using DIA PRTC retention time data to predict SRM RT, then confirming that assays are present in my samples

1) Used the PRTC peptides.sky.zip skyline doc in Skyline Daily, imported my converted DIA sample files, then exported a Peptide Retention Time report.

2) Exported Peptide Retetion Time report from my final selected transitions Skyline project

3) more TBD…

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Laura’s Notebook: SRM mass spec refresher, prepping samples with PRTC

Mass Spec refresher, final sample prep (+PRTC, transferring to autosampler vials)

Prepare PRTC from stock solution to add to samples:

• As in the DIA preparation I did the following with Peptide Retention Time Calibration Mixture ThermoFisher #88320, stock concentration is 0.5pmol/ul.
• Pipetted 20ul of PRTC into new tube – the PRTC I used was in Emma’s box, and was an aliquot of 20ul.
• Added 30ul acetonitrile, vortexed & centrifuged lightly
• Final volume is 50ul of a [200fmol/ul]
• Pipetted 1.88ul into each autosampler vial
  • Oh NO! I forgot that i needed to first pipette my mixtures into centrifuge tubes, mix, then transfer to the autsampler vial. I salvaged as much diluted PRTC as I could and restarted.
• I needed more PRTC. There was another aliquot of stock PRTC in my sample box (as per Yaamini). It was not a full 20ul. I pipetted 10 ul stock PRTC into my PRTC dilution tube, then added 15ul “Final Solvent,” vortexed then spun down. Thus, I ended up using a mix of: salvaged initial mix of PRTC + 25ul second preparation.

Prepared PRTC, Sample, and Final Solvent mix

Labeled new mini centrifuge tubes (0.75ul) and autosampler vials with my sample #’s, organized them in the order I will run them. I will add PRTC, sample & final solvent to the centrifuge tubes, mix, then transfer to the autosampler vials.

Equations from Emma to calculate volumes to add:

• How much PRTC volume should be added?
  • (PRTC final concentration)(final volume)(1/PRTC stock concentration), i.e. (25 fmol/μl)(15 μl)(1/200 fmol/μl) = 1.88ul PRTC for all my geoduck samples
• How much sample should be added to each autosampler vial?
  • ((μg to be injected/injection volume)final volume)/sample concentration, i.e. ((1 μg/2 μl)15 μl)/concentration
• How much Final Solvent to add to each autosampler vial? Final volume should be 15ul
  • 15ul – vol PRTC – vol Sample
• Calculations in “Samples for SRM” spreadsheet
• Samples G002 & G016 concentrations were low, and thus the vol sample was >15ul. In these cases I added 3.13ul sample (15ul – 1.88ul PRTC).
• Worked with the first 25 samples on my randomized list- the rest I kept in the freezer and will prep on Wednesday or Friday (pending PRTC delivery). Here’s the vol of each sample, PRTC, and Final Solvent added. Highlighted samples were low in concentration and thus required irregular volumes:

Bubbles

When I set my pipette to 15ul I consistently was getting a bubble, likely b/c I had a hard less than 15ul. So, I set my pipette to 14.8, and this helped. However, in a handful of samples a bubble got into the autosampler vial and I couldn’t pop it. So, I pulled the mixture out of the autosampler vial, returned it to the centrifuge tube, centrifuged it to remove bubbles, then re-pipetted it into the autosampler vial.

Mass Spec Refresher / notes

• Loaded my samples in the mass spec tray as per the below map. NOTE: Do not open door when mass spec is “injecting”

1	2	3	4	5	6	7	8
A	Blank	QC
B	O01	O12	O22	O113	O118
C	O26	O90	G013	G120	G047	G017	G079	G127
D	G060	G009	G002	G128	G016	G071-A	G114	G045
E	G132	G031	G012	G116	G043	G015	G040	G110
F	G008	G109	G122

• Emma prepared my methods file using the transitions I sent her and saved on the mass spec computer
• Edit the sequence table to assign sample vial location, sample & file name, vol to inject, and method to use. E.g.:
  • File name: auto-fills based on previous line; can edit if necessary. I’m sticking with Emma/Yaamini’s naming convention of “2017July10_bivalves#” where # increases sequentially for samples AND blanks.
  • Comment: sample ID, e.g. G013
  • Path: directory where data will be saved.
  • Instrument method (Inst Meth): path to the method file – Emma prepped this for me
  • Position: Tray:row,column – for example my G013 sample is located in, 1:C,3
  • Injection Volume (Inj Vol): 2ul for samples, 3ul for blank and QC

• The above image shows my sequence for the first 25 samples, starting below the highlighted cell. You’ll see a pattern: Run 5 samples, then a QC, then a blank. Repeat.
• NOTE: if anything in the Acquisition Queue needs to be edited, it’s best to 1) delete existing, incorrect sequence row in the acquisition queue, 2) create a new row in the sequence table with the correct info, 3) add to the acquisition queue.

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Laura’s Notebook: Second SRM sample prep

Readied last 25 peptide samples for mass spec

0) Labeled mini centrifuge tubes with second batch of samples, and reviewed calculated volumes of sample & Final Solvent to add to each:

1) Borrowed one last 20ul aliquot of PRTC stock from Emma/Jenn – PRO TIP: order mass spec supples way in advance! 2) Added 30ul Final Solvent, vortexted, spun down 3) Pipetted 1.88ul diluted PRTC to centrifuge tubes 4) Pipetted designated volume of Final Solvent, as per my calculates 5) Got samples out of freezer, pipetted designated vol. into centrifuge tubes as per my calculations; vortexed, centrifuged lightly. 6) Transferred mass spec-ready mixtures to autosampler vials; instead of pipetting 15ul I used 14.7, since using 15ul consistently resulted in air bubbles. Despite this, I had to re-pipette samples 54 & 55. 7) Added these newly prepared samples to the sequence table, continuing the pattern of QC, blank, 5 samples, repeat. 8) Loaded the samples on to Tray 2 in the following order:

1	2	3	4	5	6	7	8
A
B	G041	G066	G105	G032	G129	G054	G081	G003
C	G074	G014	G049	G053	G104	G055	G042	G064
D	G073	G057	G007	G070	G001	G071-B	G062
E	GBlank	OBlank
F

9) Added 65ul Final Solvent to the blank vial; there should be ~130ul blank as of 5:30pm today.

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Laura’s Notebook: Checking in on my oysters

Stopped by Manchester for the AM to check on things.

• Downwellers inside were very dirty, holding lots of water but not overflowing. I cleaned them with fresh water.
• I have 9 larval groups left (down from 16). Changed banjos. Water flow and food all looked good, no overflows.
• Water change on SN growth experiment. This week I don’t have time to check these oysters out, but next week I plan to look at them under the scope, and assess whether or not I can count live/dead easily, and how I can keep them separate to potentially extend the experiment (is it even possible?)
• Outside tank:
  • Drained tank
  • Rinsed downwellers and upwellers with fresh water. Downwellers were clean (since Olivia cleaned them on Monday), but upwellers were very dirty. After rinsing them, I uncovered some beautiful oysters! Here is the NF6 Ambient silo, which has a ton of juveniles:
• I received the 1”->1/2” reducing couplers in the mail, so I set up one more downwelling string, and moved the HL-6 Ambient & HL-6 Low 180um setting silos outside.

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