Back to the C. virginica…

Back to the C. virginica DMR analysis, plus trying to improve Platanus/Redundans running.

I started a Bismark run for the C. virginia BS-Seq data using --non_directional argument or Bismark and after hard trimming the first 16 bases off of reads as suggested by Mackenzie. This improved mapping rates significantly, from ~ 8% average to the high 20s. I uploaded the bam files to owl here and notebook here.

After finishing that, I started running the data through some methylation extraction options including bismark_methylation_extractor and MethylExtract. Having two options should allow me the ability to test a few different options for DMR calling. As of writing the Bismark extraction has finished with data available here and notebook found here

On the subject of Platanus and Redundans, I’ve been trying to get Redundans to do gap closing with the illumina short reads, but for some reason it isn’t able to extract reads from the 50bp reads. Not sure on why this is, but have been experimenting with a few different arguments in Redundans to see if that helps at all. Will update when I’ve found a solution.

Yaamini’s Notebook: DNR Sonication Round 2

How many times can you restart sample preparation?

Two, and hopefully that’s it. Before sonication, we add 50 mM NH4HCO3 + 6M urea solution to each of our samples. After we added the solution to all our samples, I realized that I made a sodium bicarbonate solution instead of an ammonium bicarbonate solution yesterday. RIP.

Our samples with sodium bicarbonate + urea were tossed in the -80ºC, and Laura and Jose correctly prepared the 50 mM NH4HCO3 + 6M urea solution. The other sample halves that were saved in the freezer were then used for protein extraction. We went through the following protocol:

Added 50 mM NH4HCO3 + 6M urea solution to samples

  • Removed samples from -80ºC and placed in wet ice. Samples remained in wet ice for the duration of the sonication process.
  • Pipetted 100 µL 50 mM NH4HCO3 + 6M urea solution into one sample tube
  • Repeated for all samples + 1 blank

Homogenized samples

  • An up-and-down motion worked better than a side-to-side motion (pounding > smushing)
  • Homogenized as much as possible
  • Resulting mixture was cloudy, difficult to see tissue fragments
  • Repeated for all samples + 1 blank

Vortexed samples

  • 3 pulses on vortex set to speed 10 (maximum speed)
  • Repeated for all samples + 1 blank

Laura realized that our lab’s sonicator just received a new sonicating tip! We decided to break in the new machine today. We set the amplitude (sonication intensity) at 50%. This was the maximum amplitude we could use without any sample spraying out.

Sonication preparation

  • Filled containers with 1000 mL nanopure water each and labelled containers “1” and “2”
  • Made an ethanol dry ice bath
  • Filled a beaker with ethanol
  • Added one piece of dry ice at a time
  • Continually added ice over the course of sonication
  • Ensured all samples needing sonication were in a wet ice bath
  • Cleaned sonicator tip
  • Dipped in ethanol for 5 seconds while on
  • Dipped in first container of nanopure water (1) for 5 seconds while on
  • Dipped in second container of nanopure water (2) for 5 seconds while on
  • Wiped down with additional ethanol and a kim wipe

Sonication

  • Sonicated one sample
  • Placed sonicator tip in one sample centrifuge tube for 10 seconds
  • Immediately placed sample in ethanol dry ice bath for 5 seconds
  • Moved sample to wet ice bath
  • Cleaned sonicator tip using procedure explained above
  • Repeated for the rest of the samples sequentially, and the blank
  • Repeated sonication procedure for all samples and a blank 3 times total
  • Sonication must proceed sequentially (one sample sonicated, then another sonicated)
  • Cannot sonicate one sample immediately after it has already been sonicated
  • MUST SONICATE REMAINING SAMPLES BEFORE RETURNING TO ONE SAMPLE

I sonicated my samples first since I had to get to class. I was only able to make it through the first round of sonication before Jose took over for me. He carried out the rest of my sample preparation, including transferring 11 µL of the sonicated sample to a new centrifuge tube. Tomorrow, we’ll do a BCA assay.

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