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

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

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

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

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

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

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

Remove RNA :

3µL RNAse from Qiagen Puregene Tissue Kit
Incubate 37°C 0h 15m 0s
Incubate On ice 0h 1m 0s

Protein precipitation

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

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

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

DNA isolation from cattle semen for long read sequencing

Abstract

Before start

Steps

Preparation of reagents

Preparation of sample

Lysis

Protein precipitation

DNA precipitation

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