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:

Low:

• 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

Ambient:

• 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