QuantSeq of Gonad Samples

[ksil91]

The 4 test QuantSeq libraries I did from the gonad RNA samples you sent me all worked well despite the low RNA quality (comparable read depth to other samples, ~88% read mapping to the transcriptome I assembled). I'm going to be doing 2 large scale library preps on the rest of my adult samples and can sequence the other 8 gonad RNA samples you sent me. Do you want to send me any more RNA samples from that experiment to sequence? I'm making one batch now and the next right after NSA so I could potentially bring the samples back with me in checked baggage.

[laurahspencer]

Great news that the RNA quality was adequate. Regarding sequencing ... Yes, of course! How many samples are you thinking?

[sr320]

Sure, if you don't mind making the libraries. Can someone remind me what experiment these are from? Are there sample IDs with the ones already sent?

[laurahspencer]

@sr320 list of samples (as described in issue #648) Unfortunately I sent her 10degC samples, not 6degC, as I thought that the gonads were more developed ... but these aren't the samples I've settled on for Hall grant (I'll be focusing on 6degC).

SN low pH

1. male - SN-10-20
2. female - SN-10-16
3. hermaphrodite - SN-10-17

SN ambient pH

4. male - SN-10-25
5. female - SN-10-31
6. hermaphrodite - SN-10-26

NF low pH

7. male - NF-10-24
8. female - NF-10-23
9. hermaphrodite - NF-10 22

NF ambient pH

10. male - NF-10-28 (not 100% male, a few oocytes)
11. female - NF-10-30
12. hermaphrodite - NF-10-26

[sr320]

So you want to send the 6 degree samples? Note the Hall could be used for this too. Remind us why you settled on 6 degree for Hall.

[laurahspencer]

Hall = I'm focusing on the OA impact only. 6 degree was considered "control" (historic winter temp), whereas 10C winer T was considered the climate stressor / simulating temperature during the Puget Sound "blob"

On Wed, Feb 28, 2018 at 12:44 PM, Steven Roberts notifications@github.com wrote:

So you want to send the 6 degree samples? Note the Hall could be used for this too. Remind us why you settled on 6 degree for Hall.

— You are receiving this because you were assigned. Reply to this email directly, view it on GitHub https://github.com/RobertsLab/project-olympia.oyster-genomic/issues/34#issuecomment-369376357, or mute the thread https://github.com/notifications/unsubscribe-auth/AQdwfX1dh7QNZwI0nYwgMAmotyUV25Uaks5tZbqjgaJpZM4SXSUC .

-- Cheers, Laura H Spencer Graduate Student School of Aquatic & Fishery Sciences University of Washington, Seattle https://laurahspencer.github.io/LabNotebook/

[ksil91]

If you don't need the samples you already sent sequenced, let me know and I just won't make libraries for them.

Up to 24 samples would be easy to do and throw in with what I have planned. More would still be fine time-wise, but would probably require another lane ($750) and potentially another kit (~$20/sample)

[sr320]

In terms of deadline - we need answers / samples by ?

On Wed, Feb 28, 2018 at 4:23 PM Katherine Silliman notifications@github.com wrote:

If you don't need the samples you already sent sequenced, let me know and I just won't make libraries for them.

Up to 24 samples would be easy to do and throw in with what I have planned. More would still be fine time-wise, but would probably require another lane ($750) and potentially another kit (~$20/sample)

— You are receiving this because you were mentioned.

Reply to this email directly, view it on GitHub https://github.com/RobertsLab/project-olympia.oyster-genomic/issues/34#issuecomment-369430832, or mute the thread https://github.com/notifications/unsubscribe-auth/AEPHt5lfLXSDQqxVhU6wF1HdGGvfwztEks5tZz80gaJpZM4SXSUC .

[sr320]

@laurahspencer Lets discuss this ASAP

[ksil91]

Preferably samples by April 6.

[laurahspencer]

@ksil91 I isolated RNA from the remaining South Sound gonad samples this week, with surprisingly good results (in terms of RNA concentration), and will extract RNA from larval samples this afternoon. How much RNA (ng) do you need per sample? I'll use that to inform which samples I select. Also, I've isolated total RNA, which is what Sam sent you previously, and I presume that's OK?

[ksil91]

Total RNA is great. Ideal samples are >80 ng/uL (at least 10 uL), but I can work with lower concentrations if total ng RNA is over 600-700.

[laurahspencer]

Great, I have both gonad and larvae RNA. I can get these ready to ship tomorrow, Thursday or Friday. @ksil91 & @kubu4 can you advise on how to prepare & ship these samples?

[kubu4]

Ship on dry ice, no later than tomorrow (assuming @ksil91 is available to receive them on Thursday). Generally, don't ship dry ice any later than Wednesdays.

Unless you've had the hazardous shipping training that encompasses dry ice shipping, I don't think you can ship these. As such, I'll take care of the paper work.

As long as you have the box all ready to go (samples and dry ice in styrofoam cooler, inside a cardboard box; taped shut) by 1:30PM, I'll get them shipped.

@ksil91 - If you confirm shipment is OK, should I use the address you've previously used for shipping to you?

[ksil91]

Shipment this week is fine, but yeah try not to ship on a Thursday or Friday just to ensure they get in on a weekday. Sam you can use the same address as usual.

How many samples, and do you want them all made into libraries? Please me a spreadsheet or sample list.

[laurahspencer]

Great, I'll have them packaged tomorrow AM to be sent off.

Katherine - I've targeted 24 samples as per your suggestion, but whatever you have capacity to do (including making libraries) works for me. I'll probably send them all to you (I have a few more than 24), then follow up with the spreadsheet / sample list in order of priority.

[kubu4]

The package is on its way via FedEx: 771916783619

[ksil91]

Package received! Send me a spreadsheet/sample list by next week (order of priority would be great). Might be good to include some info on treatments for each sample and what the research question is (so for example if somehow all samples of a treatment fail to make quality libraries we can discuss how to move forward).

These samples were also extracted from histology tissues, correct?

Also, please confirm if you do or do not want the rest of the initial gonad samples sent to be sequenced.

[laurahspencer]

Great! I'll get everything to you this weekend.

I sent you gonad rna extracted from tissue in histology blocks (samples 1-33), and rna from frozen larvae (34-47). I'm going to select 16 of the gonad samples, and 8 of the larvae samples. I may or may not select a few of the "SN" (south sound) gonad samples you already have - I need to cross-check the sex/stage of all the samples before selecting.
Thank you!

[laurahspencer]

Here is the data sheet with info for the selected samples (and a few back-ups) - I've included a few comments on the Excel file with explanations. https://github.com/laurahspencer/O.lurida_Stress/blob/master/Data/2018-04-08-QuantSeq-Samples.xlsx

For quick reference, here are the 24 samples I selected. Feel free to call tomorrow with questions!:

RNA Sample #	Original Sample Label	Temperature	pH	Gonad Sex	Gonad Stage	Tissue Type
2	SN-6-17	6	Low	HPF	2	Fixed Gonad
3	SN-6-18	6	Low	H	2	Fixed Gonad
7	SN-6-22	6	Low	H	2	Fixed Gonad
10	SN-6-25	6	Ambient	HPF	2	Fixed Gonad
11	SN-6-26	6	Ambient	F	2	Fixed Gonad
13	SN-6-28	6	Ambient	HPM	2	Fixed Gonad
14	SN-6-29	6	Ambient	HPF	3	Fixed Gonad
15	SN-6-30	6	Ambient	F	3	Fixed Gonad
n/a (Sam sent previously)	SN-10-16	10	Low	F	3	Fixed Gonad
19	SN-10-18	10	Low	HPM	2	Fixed Gonad
21	SN-10-21	10	Low	F	2	Fixed Gonad
23	SN-10-23	10	Low	H	2	Fixed Gonad
24	SN-10-24	10	Low	F	2	Fixed Gonad
29	SN-10-29	10	Ambient	F	2	Fixed Gonad
32	SN-10-32	10	Ambient	H	2	Fixed Gonad
33	SN-10-33	10	Ambient	HPF	2	Fixed Gonad
34	77-A	6	SN-6-Ambient	n/a	n/a	Frozen D-hinge larvae
35	10-A	6	SN-6-Ambient	n/a	n/a	Frozen D-hinge larvae
37	69-A	6	SN-6-Ambient	n/a	n/a	Frozen D-hinge larvae
39	48-A	6	SN-6-Ambient	n/a	n/a	Frozen D-hinge larvae
41	6-A	10	SN-10-Low	n/a	n/a	Frozen D-hinge larvae
44	32-A	10	SN-10-Low	n/a	n/a	Frozen D-hinge larvae
45	79-A	10	SN-10-Low	n/a	n/a	Frozen D-hinge larvae
47	26-A	10	SN-10-Low	n/a	n/a	Frozen D-hinge larvae