DNA isolation from cattle semen for long read sequencing

Cecile Grohs, Erwan Denis, Carole Iampietro

Published: 2024-01-18 DOI: 10.17504/protocols.io.j8nlkw1qwl5r/v1

extraction

high molecular weight

sperm

DNA

Long read sequencing

bovine

DNA isolation

PacBio

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Abstract

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.

Steps

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:

-450µL

-50µL

Note
This mixture of a guanidine-based reagent (RLT) and a thiol-free reducing agent facilitate dissociation of disulfide bonds (Wu et al , 2018).TCEP is odorless, and more stable than DTT (Han & Han, 1994).

Citation
Han JC & Han GY 1994 A Procedure for Quantitative Determination of Tris(2-Carboxyethyl)phosphine, an Odorless Reducing Agent More Stable and Effective Than Dithiothreitol Analytical Biochemistry https://doi.org/10.1006/abio.1994.1290

Citation
Wu H, de Gannes MK, Luchetti G, Pilsner JR 2015 Rapid method for the isolation of mammalian sperm DNA. https://doi.org/10.2144/000114280

Preparation of sample

2.

Recovery of spermatozoa from the straw:

  • Empty the 200µL``Sample in a 2mL by cutting the two ends of the straw
  • Rince the straw it with 200µL Room temperature
3.

Wash:

  • Add 800µL more PBS (up to 1mL )

  • Pellet 1000x g

  • Discard the supernatant

Second wash is optional (no significant impact observed)

-Re-suspend in 1mL

-Pellet 1000x g

-Discard the supernatant

Note
Centrifuge gently so that the pellet does not stick. It should be easy to resuspend for efficient lysis.

Lysis

4.

Step one:

Add 500µL of RLT-TCEP to the pellet

  • Vortex 0h 0m 10s by pulsing at max speed

  • If necessary, use a wide opening tip to resuspend the pellet

  • Incubate On ice 0h 10m 0s

5.

Step two: continue with Qiagen Puregene Tissue kit adapted as follow

  • Add 500µL of Cell Lysis Solution

  • Add 60µL of 20mg/mL proteinase K (20 mg/ml)

  • Mix by inversion ( about 25 inversions)

  • Incubate 55°C 1h 30m 0s

6.

Remove RNA :

  • 3µL RNAse from Qiagen Puregene Tissue Kit

  • Incubate 37°C 0h 15m 0s

  • Incubate On ice 0h 1m 0s

Protein precipitation

7.

  • Add 200µL of Protein precipitation buffer (from Qiagen Puregene Tissue Kit)

  • Mix by hand or gently vortexing 0h 0m 15s

  • Incubate On ice 0h 5m 0s

  • Centrifuge 16000x g

DNA precipitation

8.

  • Transfert the supernatant to a new tube containing 600µL of Isopropanol

  • Carrefully invert the tube 25-50X times to form the pellet

  • Incubate 0h 5m 0s``Room temperature

  • Centrifuge 16000x g

  • Discard supernatant

9.

  • Add 600µL of 70% ethanol to the pellet

  • Centrifuge 5000x g

  • Discard supernatant

  • Almost dry the pellet Room temperature``0h 5m 0s

  • Add 50µL to 100µL of EB (Qiagen) or TE buffer to eluate DNA

  • Store DNA at 4°C

Note
DNA in EB buffer can be heated to 60°C for 1 hour to dissolve it. Do not vortex or pipet DNA.It is recommended not to freeze the DNA to preserve long fragments.

Citation
Of the 60 extractions carried out using this protocol, the average size of the fragments generated is around 53 kb, ranging from 25 to 120 kb on average. We expect 30 ug of DNA from a commercial semen straw, but this figure can vary considerably from sample to sample. We obtained absorbance ratios of 260/280 for DNA of around 1.8 nm, and 260/230 ratios averaging 0.5 nm. Low ratios have already been observed using RLT buffer (Wu et al , 2018), but these did not affect PacBio sequencing significantly.Some of these DNA have been sequenced and published in Jourdain et al. 2023.

Citation
Jourdain J, Barasc H, Faraut T, Calgaro A, Bonnet N, Marcuzzo C, Suin A, Barbat A, Hozé C, Besnard F, Taussat S, Grohs C, Kuchly C, Iampietro C, Donnadieu C, Pinton A, Boichard D, Capitan A 2023 Large-scale detection and characterization of interchromosomal rearrangements in normozoospermic bulls using massive genotype and phenotype data sets. https://doi.org/10.1101/gr.277787.123

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