Lots of time in the lab this last week making 70 QuantSeq mRNA libraries from Olympia oyster RNA samples during the rangewide low pH experiment. Submitted to the sequencing core today! You can see the lab notebook for these libraries, along with extraction and library prep protocol here.
Today I submitted two QuantSeq 3′ mRNA-Seq libraries to the UChicago Functional Genomics Core for sequencing. Each library has 28 samples and will be sequenced on a separate 50bp SE HiSeq 4000 lane. For one library, I quantified the finished PCR products for each sample using Qubit and checked the quality using Bioanalyzer HS before pooling them myself. For the other library, I am letting the sequencing center pool the samples by estimating concentrations using qPCR. This is more expensive per sample (and slows down decision making as I’m not doing the QC myself), so I want to compare the two libraries and see if I can achieve fairly even sequencing with just Qubit. You can see my lab notebook and protocols here.
Today submitted RNA for rock scallops (Crassodoma gigantea) for preparation into mRNA directional libraries and sequencing. Details on the RNA extractions and QC can be found here.
Update on July goals:
- I’m just waiting on a comments from a couple of people, but otherwise the Puget Sound common garden paper is written and ready to go
- I decided to use a mollusc-specific kit for RNA extractions from the abductor muscle because the QC/concentration kept being so low. Kit is now in and anticipating scallops will be sent off for sequencing next week.
- Got final confirmation that Mark and I won’t be able to outsource preparation of our TagSeq libraries at UT, so we will be buying a small kit and making test libraries this month.
- Waiting until August 7 or so to send probes off, as I want to check out one other potential transcriptome resource.
Little late to post, but whiteboard goals for July 2017!
For the past 3 weeks I’ve been hanging around the Pacific Northwest, first going out to Tatoosh Island with my adviser, followed with some hiking, and then finishing up at the Evolution meeting in Portland, OR. I gave an oral presentation of the first chapter of my dissertation on the population structure of Olympia oysters that you can check out here, and a poster of a paper that’s going to be submitted soon looking at adaptive differences among 3 populations of oysters in Puget Sound, WA (here). I’ve also been cleaning up my Github so that scripts and notebooks are better organized by project and have more documentation to them. In particular, I cleaned up the notebooks detailing the figures I made in my talk at Evolution.
I did some initial RNA extractions on scallop tissues in preparation for preparing a transcriptome. A lot of them worked pretty well! See the lab notes and protocol here.
I submitted another 2bRAD library today. This one has 25 samples of pooled Ostrea lurida larvae (and 3 samples from juvenile spat). Some of the samples correspond to the growth experiments done in Summer 2015, some of them correspond to the G2 South Sound oysters being used in Laura’s experiment, and some are just extras. The main goals of this library are to:
- See what pooled 2bRAD data “looks” like
- determine the amount of sequencing depth needed for confident genotyping (i.e. how many samples can go on a lane)
- determine if parentage can be assigned using 2bRAD sequencing from adults
- Bonus if successful- get an idea of how many parents contributed to my experiments
Details and notes from the library prep (including protocol) can be found at this Benchling note.
Yesterday I submitted a 2bRAD library for sequencing at the UChicago Genomics Core, with an anticipated turn-around time of 1-2 weeks. The details of the library prep can be found at this Benchling note. At the top of the note there is also a tab for the protocol I followed, with the specific master mixes made for this library (one of my favorite features of Benchling).
I had my Fall 2016 committee meeting on Thursday, Dec. 8. Pretty productive, with some good discussion about what to do going forward with my samples from this summer’s field season. Slides can be found here.