WGBS Samples Part 4

More planning for WGBS

After figuring out which samples to sequence, I wanted to confirm I didn’t need to do anything besides have 500 ng DNA at 20 ng/µL. In this issue, I confirmed I wouldn’t have to shear DNA! My next step was checking DNA concentrations. First, I measured the volume of the DNA samples. Then, I checked concentrations with the Qubit. When I compared the concentrations I got when I initially extracted the DNA in this table, I noticed that some samples had drastically different concentrations (06-T1, UK-01, UK-02, UK-03, UK-04). I ran these samples again, along with two others (UK-07, UK-08). I wanted to see if these two samples, which were from females at similar maturation stages, also had different DNA concentrations and yields that would make them usable for WGBS:

Table 1. Abbreviated sample volumes and revised concentrations. If a sample will be used for sequencing, I indicated whether or not I need to do an ethanol precipitation (Yes/No). If the total DNA in a sample is < 400 ng, I decided not to use it and added “N/A” in the ethanol precipitation column. Run 1: S1 = 233.79, S2 = 26196.20. Run 2: S1 = 286.00, S2 = 34707.88.

Sample Treatment Volume (µL) Concentration 1 (ng/µL) Concentration 2 (ng/µL) Ethanol Precipitation?
02-T1 Low 40 37 N/A No
04-T3 Low 40 20.8 N/A No
06-T1 Low 40 47.2 44.6 No
07-T2 Low 40 15.4 N/A Yes
08-T2 Low 40 22.2 N/A No
10-T3 Low 20 23.2 N/A N/A
11-T4 Ambient 30 17.6 N/A Yes
UK-01 Ambient 35 9.16 8.84 N/A
UK-02 Ambient 45 10.9 11.7 Yes
UK-03 Ambient 40 12.4 10.9 Yes
UK-04 Ambient 40 66.8 31.6 No
UK-06 Ambient 45 19.6 N/A Yes
UK-07 Ambient 40 N/A 5.86 N/A
UK-08 Ambient 40 N/A 8.02 N/A

I added these results to my spreadsheet and calculated a conservative total DNA yield in each sample based on the lowest DNA concentration I got today. From those concentrations, calculated yields, and balancing sex and maturation stage, I narrowed down my list to 10 samples. I added the information for possible RNA extractions found in this noteboo post for reference:


  • 02-T1
  • 04-T3
  • 06-T1: No tissue left for RNA extraction
  • 07-T2
  • 08-T2: Less than half of tissue sample left for RNA extraction


  • 11-T4
  • UK-02: Less than 500 ng
  • UK-03: Less than 500 ng
  • UK-04
  • UK-06: Less than half of tissue sample left for RNA extraction

Going forward

  1. Prepare dilutions and run them on the BioAnalyzer
  2. Complete isopropanol precipitations
  3. Extract RNA from gonad samples
Written on July 31, 2020