Yaamini’s Notebook: DML Analysis Part 10

Testing bismark parameters

After meeting with Steven this week, we decided the best course of action was to revise the analysis pipeline and document why we chose the settings we did. The first part of this revision is to go back to bismark. When I ran my full samples through bismark, I used the default setting for -score_min. This lead to a ~20% mapping efficiency. That’s not great.

The -score_min option dictates how strict the alignment is. The default option is L,0,-0.2, which is pretty stringent. In this Jupyter notebook, I tested 3 different -score_min options: L,0,-0.6; L,0,-0.9; L,0,-1.2. Samples were run in the following order:

screen shot 2018-10-04 at 10 47 55 am

Therefore, the first mapping efficiency listed belongs to sample 10, second to sample 1, etc. I kept this in mind and generated the following table:

Table 1. Mapping efficiency (%) based on different -score_min settings.

Sample L,0,-0.6 L,0,-0.9 L,0,-1.2
1 15.5 20.2 28.8
2 32.4 40.2 49.8
3 37.2 45.3 53.6
4 36.0 44.7 52.9
5 34.6 42.9 51.7
6 36.7 45.0 53.8
7 34.6 42.9 51.4
8 31.7 39.0 47.6
9 33.0 41.2 49.9
10 36.6 44.9 53.0

To optimize mapping efficiency, we decided to run full samples with -score_min L,0,-1.2. I started the analysis in a new Jupyter notebook.

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Ronit’s Notebook: RNA Extraction for C.Gigas Heat Stress Samples

I started working with the C. Gigas heat stress samples to extract RNA for gene expression analysis. Sam took care of 8 of the samples and I did the RNA extraction for the other 8 samples: D09, D10, D11, D12, T09, T10, T11, T12

The protocol I followed is as follows:

  1. 500 µL of RNAzol RT was added to a clean tube.
  2. Tissue samples were removed and a small section was cut out for RNA extraction.
  3. Tissue portions were placed in the tube and an additional 500 µL of RNAzol RT was added to bring the volume up to 1mL.
  4. The samples were vortexed vigorously for 10 seconds
  5. Samples were incubated at room temperature for 5 minutes.
  6. 400 µL of DEPC-water was added to the samples.
  7. Samples were centrifuged for 15 minutes at 12,000 g.
  8. 750  µL of the supernatant was transferred to a new, clean tube and an equal volume of isopropanol was added to the sample.
  9. The samples were vortexed vigorously for 10 seconds.
  10. Samples were incubated at room temperature for 5 minutes.
  11. Samples were centrifuged for 15 minutes at 12,000 g.
  12. The supernatant was discarded and 400 µL of 75% ethanol was added to the samples.
  13. Samples were centrifuged for 1.5 minutes at 4,000 g.
  14. Supernatant was discarded and an additional 400 µL of 75% ethanol was added to the samples.
  15. Samples were stored in -80 degree freezer.

Due to time constraints, we decided to finish up the extraction next week/quantify RNA using the Qubit and stored the RNA pellet suspended in ethanol in the -80 freezer.


Sam’s Notebook: RNA Isolation – Ronit’s C.gigas diploid/triploid dessication/heat shock ctenidia tissues


Isolated RNA from a subset of Ronit’s Crassostrea gigas ctenidia samples (see Ronit’s notebook for experiment deets):

  • D01 C
  • D02 C
  • D19 C
  • D20 C
  • T01 C
  • T02 C
  • T19 C
  • T20 C

RNA was isolated using RNAzol RT (Molecular Research Center) in the following way:

  • Unweighed, frozen tissue transferred to 500uL of RNAzol RT and immediately homogenized with disposable pestle.
  • Added additional 500uL of RNAzol RT and vortexed to mix.
  • Added 400uL of 0.1% DEPC-treated H2O, vortexed and incubated 15mins at RT.
  • Centrifuged 12,000g for 15mins at RT.
  • Transferred 750uL of supernatant to clean tube (discarded remainder), added 1 volume (750uL) of isopropanol, vortexed, and incubated at RT for 10mins.
  • Centrifuged 12,000g for 10mins at RT.
  • Discarded supernatant.
  • Washed pellet with 75% ethanol (made with 0.1% DEPC-treated H2O).
  • Centrifuged 4,000g for 2mins at RT.
  • Discarded supernatant and repeated wash steps.

Pellet was resuspended in 50uL of 0.1% DEPC-treated H2O and stored @ -80oC in Ronit’s temporary box.

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