Goals – August 2015

Review of last month’s goals:

O.lurida RNA Isolation & Reverse Transcription -

RNA has been isolated and DNased from Jake’s mechanically stressed samples.

Miscellany -

Improved organization of -80C, added frequently used protocols to the Roberts Lab GitHub Wiki, connected BGI rep with purchasing to get the PO situation figured out for Olympia oyster and geoduck genome sequencing, sent samples to BGI for Olympia oyster genome-by-sequencing (GBS).

 O.lurida Reverse Transcription

Need to quantify the DNased RNA from Jake’s mechanically stressed oysters and then verify that the DNase treatment worked. Will then proceed with reverse transcription.

Miscellany

Continue work on -80C organization, continue creating “readme” files for folders on our server(s), continue migration from Wikispaces to GitHub, attempt to combine our PrimerDatabase and our Primer Stocks spreadsheets into a single document (and create a SQL database from that), fix the shortfalls from our EH&S lab inspection.

Server HDD Failure – Owl

We had our first true test of the Synology RAID redundancy with our Synology 1812+ server (Owl). One of the hard drives (HDD) failed. All of the other drives were fine, the data was intact and we had a new replacement HDD on hand. However, there was one shortcoming: no email notification of the drive failure. Luckily, the Synology server is next to Steven’s office and he could hear an audible beeping alerting him to the fact that something was wrong. In any case, the email notifications have been fixed and a replacement hard drive was added to the system. Here’s how these things were accomplished.

 

Fix email notifications

The system was previously set to use Steven’s Comcast SMTP server. Sending a test email from Owl failed, indicating authentication failure. I changed this to use the University of Washington’s email server for outgoing messages. Here’s how…

In the Synology Disk Station Manager (DSM):

Control Panel > Notifications

  • Service provider: Custom SMTP Server
  • SMTP server: smtp.washington.edu
  • SMTP port: 587
  • Username: myUWnetID@uw.edu
  • Password: myUWpassword

Interesting note, there’s a “Push Service” tab in the “Notifications” window. This allows you to have Synology send emails to email addresses when the server has an issue. This eliminates the need for the SMTP settings shown above which may not be easy to find and/or understand for a given email service provider. The “Push Service” appears to be much simpler and more user friendly to set up.

 

Hot Swap HDD

We’ve kept a backup HDD on hand for just this occasion, so the HDD failure wasn’t too concerning. Here’re the steps I followed to swap the HDD and have the Synology system initialize/build the new HDD:

 

Remove the dead HDD and put the new HDD in.

 

 

Initialize/build/repair the new HDD.

In Synology DSM:

Storage Manger > Volume

Notice, there should be eight drives listed, but since one has died, only seven are shown:

 

 

 

 

 

 

 

That’s it! Easy breezy!

I’ve checked with Seagate on the dead HDD and it is still under warranty. Will get that returned and also purchase a new backup drive to have on hand.

DNase Treatment – O.lurida Ctenidia 1hr Post-Mechanical Stress RNA

Quantified the RNA I isolated from Jake’s samples on 20150715 and 20150710 using the Roberts Lab NanoDrop1000 (ThermoFisher).

 

 

 

Overall, the yields are good. The 260/280 ratios are mediocre. Will proceed with DNase treatment.

DNased 1.5ug of RNA from each sample using the Turbo DNA-free Kit (Ambion/Life Technologies), following the “rigorous” protocol.

Briefly:

  • 50μL reactions were carried out in 0.5mL tubes
  • added 1μL of DNase to each tube
  • incubated 30mins @ 37C
  • added additional 1μL of DNased
  • incubated 30mins @ 37C
  • added 0.2 vols (10.2μL) of DNase Inactivation Reagent
  • incubated and mixed for 2mins @ RT
  • spun 1.5mins, 10,000g @ RT
  • transferred 50μL of supe to sterile 1.5mL snap cap tubes
  • spec’d on Roberts Lab NanoDrop1000

Samples were stored @ -80C in Shellfish RNA Box #6. Will quantify at a later date.

DNase reaction calcs: 20150727_Jake_Oly_mech_stress_DNase_calcs

 

RNA Isolation – O.lurida Ctenidia 1hr Post-Mechanical Stress

Isolated RNA from Jake’s Ostrea lurida ctenidia samples that had been subjected to mechanical stress (from 20150422).

Despite the indication in this notebook, the samples had not been previously homogenized in RNAzol RT. I thawed the samples, homogenized them and followed the RNAzol RT protocol for total RNA isolation. Here’s the list of samples:

  • 42215 HM1 1
  • 42215 HM1 2
  • 42215 HM1 3
  • 42215 HM1 4
  • 42215 HM1 5
  • 42215 HM1 6
  • 42215 HM1 7
  • 42215 HM1 8
  • 42215 SM1 1
  • 42215 SM1 2
  • 42215 SM1 3
  • 42215 SM1 4
  • 42215 SM1 5
  • 42215 SM1 6
  • 42215 SM1 7
  • 42215 SM1 8

RNA was resuspended in 50μL of 0.1%DEPC-H2O and stored @ -80C in the original box they came from.

RNA Isolation – O.lurida Ctenidia 1hr Post-Mechanical Stress

Isolated RNA from Jake’s Ostrea lurida ctenidia samples that had been subjected to mechanical stress (from 20150422).

Despite the indication in this notebook, the samples had not been previously homogenized in RNAzol RT. I thawed the samples, homogenized them and followed the RNAzol RT protocol for total RNA isolation. Here’s the list of samples:

  • 42215 NM1 1
  • 42215 NM1 2
  • 42215 NM1 3
  • 42215 NM1 4
  • 42215 NM1 5
  • 42215 NM1 6
  • 42215 NM1 7
  • 42215 NM1 8

RNA was resuspended in 50μL of 0.1%DEPC-H2O and stored @ -80C in the original box they came from.

Automatic Notebook Backups – wget Script & Synology Task Scheduler

UPDATE 20150714 – READ ENTIRE POST

I’ve been tweaking a shell script (notebook_backups.sh) to use the shell program wget to retrieve fully functional HTML versions of our online notebooks for offline viewing. I had been planning on setting up a cron job to automatically run this script on our Synology server (Eagle) at a set day/time. However, I came across the Task Scheduler that’s built right into the Synology GUI! So, I set up the Task Scheduler to run the notebook_backups.sh script every Sunday. See screenshots below.

 

 

 

UPDATE 201507114

The Task Scheduler was not running the script. Additionally, the Task Scheduler would not run the script even when I manually instructed the Task Scheduler to run. Some internet searching revealed that the Task Scheduler requires you to indicate what type of task is being run (e.g. bash, shell, ash, php, etc.), even if your script contains the proper “shebang” or header that normally instructs the computer which program to use to run the script. See the image below for how the Task Scheduler is currently set up. The arrow indicates that addition of “sh” to the beginning of the Task Scheduler’s path to the script. This tells the Task Scheduler to use the Shell to run the script.

 

PCR – Sea Pen luciferase

Ran a PCR to obtain luciferase DNA for sequencing.

Used sea pen gDNA extracted by Jonathan on 20150527.

Primers:

SRID Name
1604 Rr_46_65F
1603 Rr_887_868R

Master mix calcs are here: 20150702_seapen_PCR

Cycling params:

  1. 95C – 10mins
  2. 95C – 15s
  3. 55C – 15s
  4. 72C – 1min
  5. Go to Step 2 39 times

 

Ran samples on 0.8% agarose, low TAE gel stained with EtBr.

Results:

 

 

Loading:

Lane 1 – ladder

Lane 2 – empty

Lane 3 – sea pen gDNA

Lane 4 – NTC

 

PCR did not work. Was expecting a band of ~800bp.

Looks like I may have overloaded the PCR reaction with gDNA. Used 10μL of gDNA.

However, that is quite the smear, suggesting a significant amount of degradation present in the gDNA.

Will re-run this PCR next week with less gDNA (or, cDNA instead) in order to generate a PCR product.

GOALS – July 2015

Before we check out this month’s goals, let’s have a quick review of last month’s goals and which, if any, I was able to accomplish:

 From June 2015:

Geoduck Reproductive Development Transcriptomics

Done. Data from this was received 20150629.

BS-Seq Illumina Data Assembly/Mapping – C.gigas larvae OA

Little progress. No response from Univ. of Oregon. However, it seems that Mac has been having a similar issue with libraries constructed using Epigentek kits. She has contacted Illumina and Epigentek for help.

Geoduck & Olympia Oyster Genome Sequencing

No progress. Purchasing personnel (both departmental and university) appear to have had difficulty contacting BGI. I have put both purchasing personnel in contact with the BGI rep (Frank Hu), so things are starting to progress. Still not certain why they were unable to accomplish this.

Miscellany

Goal(s):

  • Migrate Wikispaces notebook to this notebook
  • Add to GitHub code pages
  • Flesh out/create README files on server(s)
  • Lab cleanup tasks
  • Assist on Jonathan’s Capstone project
  • Establish go-to location for lab personnel questions
  • Teach Unix shell at Software Carpentry

Status:

  • Migrate Wikispaces notebook to this notebook – Minimal progress
  • Add to GitHub code pages – Updated “Commercial Protocols”, improved wget command for offline notebook backups
  • Flesh out/create README files on server(s) – Some progress, but mostly on the notebook backups and newly received transcriptomic data. No progress on older directories.
  • Lab cleanup tasks – Partial progress. Autoclaved multiple bags of old autoclave trash.
  • Assist on Jonathan’s Capstone project – Improved my time management working with Jonathan, but his sequencing results are a bit odd and he needs guidance on how to manipulate, and comprehend, what the data means.
  • Successfully taught both the shell and GitHub at Software Carpentry.

 

O.lurida RNA Isolation and Reverse Transcription

Need to isolate RNA from a set of Jake’s mechanically-stressed oyster tissue. Need to clarify with Steven if we want the 1hr post-mechanically stressed to “match” with the 1hr heat shock samples that were previously processed, or if we want the 24hr post-mechanically stressed samples.

Miscellany

Need to address short-falls from the EH&S annual lab inspection. Most are minor, easily addressed issues and won’t take much time (e.g. print lab safety signs in color). The various computer tasks still stand: notebook migration from Wikispaces to this notebook, transitioning lab resources from Wikispaces to GitHub, creating/updating “readme” files for directories on our servers, etc. Lab organization goals: improve -80C organization and create an online inventory of the -80C, establish an online inventory of lab supply locations (i.e. thermometers can be found in FTR209, Drawer #02), general clean up.