TITLE: DNA isolation from cattle semen  for long read sequencing
AUTHORS: Erwan Denis, Cecile CG Grohs, Carole Iampietro

[DESCRIPTION]
Here we describe a method for isolate high molecular weight DNA from commercially available frozen bull semen straws.
This protocol is based on a salting-out method and uses several commercially available solutions. It consists of several steps: washing of semen, lysis, removal of proteins and precipitation of genomic DNA.
This protocol was used to isolate DNA from sixty semen straws, all of which were successfully sequenced using the CLR sequencing mode on the PacBio SequelII platform.



[BEFORE_START]
As we use commercial sperm straws to perform our extractions, we do not always know the composition of these straws, the quantity of material contained, the nature of the diluents and preservatives used. This is why it is sometimes necessary to use several straws to obtain enough material for sequencing. It is also sometimes wise to perform several washes (see step 3) to eliminate contaminants from diluents and preservatives.

[GUIDELINES]
Salting out is a good method to obtain high molecular weight (HMW) DNA, as it avoids damaging steps such as the use of purification columns or heavy mixing with phenol/chloroform.
Note that all mixing steps should be gentle to obtain HMW DNA fragments (from lysis steps to DNA precipitation). We also recommend to use DNA low bind tubes.

[STEPS]
SECTION: Preparation of sample
2. Recovery of spermatozoa from the straw:
- Empty the 200 µL  in a 2 mL by cutting the two ends of the straw

  

- Rince the straw it with 200 µL  Room temperature

                                                                                                                                                                                                                                                                                                            

SECTION: Lysis
4. Step one: 

Add 500 µL of RLT-TCEP to the pellet
- Vortex 10 s by pulsing at max speed
- If necessary, use a wide opening tip to resuspend the pellet
- Incubate on ice 10 min 
SECTION: Preparation of sample
3. Wash:
- Add 800 µL more PBS (up to 1 mL )
- Pellet 1000 x g, 5 min
- Discard the supernatant

Second wash is optional (no significant impact observed)
-Re-suspend in 1 mL 
-Pellet 1000 x g, 5 min
-Discard the supernatant

 


SECTION: Lysis
5. Step two: continue with Qiagen Puregene Tissue kit adapted as follow
- Add 500 µL of Cell Lysis Solution
- Add 60 µL of 20 mg/mL proteinase K (20 mg/ml)
- Mix by inversion ( about 25 inversions)
- Incubate 55 °C 90 min

 
  

SECTION: Protein precipitation
7. - Add 200 µL of Protein precipitation buffer (from Qiagen Puregene Tissue Kit)
- Mix by hand or gently vortexing 15 s 
- Incubate on ice 5 min 
- Centrifuge 16000 x g, 1 min 

SECTION: Lysis
6. Remove RNA: 
- 3 µL RNAse from Qiagen Puregene Tissue Kit
- Incubate 37 °C 15 min 
- Incubate on ice 1 min 
SECTION: Preparation of reagents
1. Immidiately before use, prepare a mix containing RLT buffer (Qiagen) and TCEP [Tris(2-carboxyethyl)phosphine hydrochloride] to a final volume of 500µL per sample as follow:
-450 µL 
-50 µL 

  
   

 

 

 


SECTION: DNA precipitation
9. - Add  600 µL of 70% ethanol to the pellet
- Centrifuge 5000 x g, 2 min 
- Discard supernatant
- Almost dry the pellet Room temperature5 min
- Add 50 µL to 100 µL of EB (Qiagen) or TE buffer to eluate DNA
- Store DNA at 4°C

  

 

 

 


SECTION: DNA precipitation
8. - Transfert the supernatant to a new tube containing 600 µL of Isopropanol
- Carrefully invert the tube 25-50X times to form the pellet
- Incubate 5 minRoom temperature 
- Centrifuge 16000 x g, 1 min 
- Discard supernatant