Some information I’ve missed
I met with Steven on Tuesday, and he suggested I do a few things:
- Figure out if the mRNA genome feature file overlaps with introns and exons
- Count the number of unique genes the gene background overlapped with and get Uniprot codes
Feature file overlaps
TL;DR: Yes, the mRNA feature file includes introns and exons.
Figure 1. Various genome feature files in IGV.
I opened the tracks in IGV and found they overlapped. I have to consider this as I think about what the overlaps between DML and DMR and exon, intron, or mRNA coding regions actually mean. My guess is that I need to consider exon and intron overlaps as a subset of the mRNA overlaps. Unless the mRNA coding region file has information that isn’t an intron or exon, I could just compare exon and intron overlaps instead of using mRNA overlaps.
Unique genes from gene background-mRNA overlaps
I went back to my R Markdown file and subsetted unique Genbank IDs from the file with gene background-mRNA overlaps and Uniprot codes. I used the following code:
uniqueBackgroundmRNAblast <- subset(backgroundmRNAblast, !duplicated(backgroundmRNAblast$Genbank)) #Subset the unique Genbank IDs from backgroundmRNAUniprot and save as a new dataframe. nrow(uniqueBackgroundmRNAblast$Genbank) #Count the number of unique genes
The gene background overlapped with 14,943 unique genes. I saved the subsetted information in this file.
- Describe gene products for all remaining DML and DMR overlaps
- Compare genes with hypermethylated vs. hypomethylated loci and regions
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Continuing sperm DNA extractions
Yesterday I finished extracting DNA from two sperm samples. Since my yields were good, I’m finishing up extractions!
Step 1. Obtain liquid nitrogen (LN2), dry ice, a small ceramic mortar and pestle, and a wide spatula. Place DNA samples in dry ice. Set a heat block to 37ºC.
Step 2. Pour LN2 into the ceramic mortar, and additional LN2 into a styrofoam cooler. Place the pestle and spatula in the cooler. While waiting for the LN2 to boil off the mortar, prepare a 10% bleach solution.
Step 3. Once the LN2 has boiled off, transfer the DNA sample (no more than 30 mg) into the mortar. Break the frozen sample with the spatula, then transfer into the mortar. If the sample does not break, slightly thaw it with heat from your hand.
- I lost a chunk of sample 48s (close to half the sample) while I was breaking it apart.
Step 4. Pulverize the DNA sample with the LN2-cooled pestle. Transfer the powder to a clean, labeled 1.5 mL microcentrifuge tube.
Step 5. Obtain a new mortar and pestle and repeat Steps 1-4 with remaining samples.
- It was only after I processed my third sample that I confirmed with Sam that I needed a new mortar and pestle each time! I processed samples 6s and 7s with the same mortar and pestle. Thankfully they are both from the same treatment (high pCO2). Between these samples, I wiped the mortar and pestle, cleaned with a 10% bleach solution, and wiped the surfaces with a clean kim wipe. After processing sample 7s, I found that some of the bleach had frozen onto the LN2-cooled pestle. I got a new mortar and pestle and processed sample 23s, after which I checked my methods with Sam.
Step 6. Obtain the E.Z.N.A. Mollusc Kit. Add 350 µL ML1 Buffer and 25 µL Proteinase K Solution to each sample. Vortex thoroughly.
Step 7. Place the samples on 37ºC heat block for overnight incubation.
Step 8. Soak used mortars and pestles in a 10% bleach solution for 5 minutes. Clean the equipment with a sponge and rinse with DI water before drying. The equipment can be sprayed with 100% ethanol to speed up the drying process.
- Friday: Finish isolating DNA and quantify with the Qubit
- Figure out where to send samples for WGBS and prepare samples accordingly
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