Yaamini’s Notebook: Sperm DNA Extractions

Extracting DNA from C. virginica sperm samples

We got C. virginica sperm and mantle samples from the Lotterhos Lab’s 2018 experiment! I’m going to use sperm samples to examine potential for epigenetic inheritance in this species. But first, I need to see if I can get usable DNA from these samples.

Sam suggested I use the E.Z.N.A Mollusc Kit for these extractions instead of DNAzol like Claire did with her sperm samples. I’m going to test this method on two sperm samples: L18A0012s and L18A0031s.

Methods

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.

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 the E.Z.N.A. Mollusc Kit. Add 350 µL ML1 Buffer and 25 µL Proteinase K Solution to each sample. Vortex thoroughly.

Step 6. Place the samples on 37ºC heat block for overnight incubation.

Going forward

  1. Wednesday: Finish isolating DNA and quantify with the Qubit. If successful, pulverize remaining samples and start overnight incubation
  2. Thursday: Finish isolating DNA from all samples and quantify with the Qubit

// Please enable JavaScript to view the comments powered by Disqus.

from the responsible grad student https://ift.tt/2Th84A3
via IFTTT

Yaamini’s Notebook: DML Analysis Part 23

DMR-mRNA gene product information

Since I didn’t get much information from my previous gene enrichment, I thought I would start describing the function of coding regions with DMRs in them. When I looked at the DMR-mRNA overlap file, I saw that there was gene product information buried in the last column:

 ID=rna48;Parent=gene35;Dbxref=GeneID:111114201,Genbank:XM_022452489.1;Name=XM_022452489.1;Note=The sequence of the model RefSeq transcript was modified relative to this genomic sequence to represent the inferred CDS: inserted 2 bases in 2 codons;exception=unclassified transcription discrepancy;gbkey=mRNA;gene=LOC111114201;model_evidence=Supporting evidence includes similarity to: 4 Proteins%2C and 99%25 coverage of the annotated genomic feature by RNAseq alignments%2C including 9 samples with support for all annotated introns;product=vacuolar protein sorting-associated protein 13B-like;transcript_id=XM_022452489.1  

My first step was to isolate the product= information from this column. The easiest thing to do would be to use awk to separate the text into multiple columns based on a ; delimiter. However, there are different numbers of ; in each line, so the product informaiton would not consistently be in the same column throughout the document. I wanted to separate out all product information, either by creating a custom delimiter or extracting all information after product=. I figured there’d be an elegant way to do this with bash, so I posted this issue. Unfortunately Sam couldn’t help me with a solution! I took the easy way out and used Excel.

I created a duplicate column, replaced product= with >, then used > as a delimiter to separate product information from the rest of the column. I then used ; as a delimiter with the column I just generated to separate the product information from the transcript ID. Finally, I deleted columns without product information that I generated throughout this process. My final document, found here, looks like this:

screen shot 2019-02-25 at 4 14 47 pm

I know you can use sed for a find-and-replace to do something similar in bash, but I couldn’t get that to work:

screen shot 2019-02-25 at 4 02 13 pm

Now that I had the product information isolated, I wanted to create a new document with summary information (gene product, percent methylation difference, number of transcript variants the DMR was found in). I figured this summary document would be a good starting place when I start writing. The summary document can be found here. DMRs were found in interesting genes, including calcium uptake (could help with calcification), cilia and flagella associated protein (sperm motility, cellular structure), cytochrome P450 (oxidative stress protein), tubulin-specific chaperones (motility-related), telomere-associated protein (could be related to cell replication and apoptosis), sperm-tail PG-rich repeat-containing protein (no idea what this does but my guess is that it’s sperm-related), and zygotic DNA replication. There were also 37 uncharacterized gene products.

Going forward

  1. Repeat this process with DMR-exon, DMR-intron, and DMR-TE overlaps
  2. Visualize gene product information
  3. Relate this information to gene enrichment

// Please enable JavaScript to view the comments powered by Disqus.

from the responsible grad student https://ift.tt/2tNxsiN
via IFTTT