# DNA isolation from cattle semen for long read sequencing v1

Erwan Denis, Cécile Grohs, Carole Iampietro

<figure><img src="image_1.png" /></figure>

To cite this version:

Erwan Denis, Cécile Grohs, Carole Iampietro. DNA isolation from cattle semen for long read sequencing v1. 2024, 10.17504/protocols.io.j8nlkw1qwl5r/v1. hal-04414012

HAL Id: hal-04414012

https://hal.inrae.fr/hal-04414012

Submitted on 24 Jan 2024

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archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
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abroad, or from public or private research centers.

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<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# DNA isolation from cattle semen for long read sequencing v1

Erwan Denis, Cécile Grohs, Carole Iampietro

► To cite this version:

Erwan Denis, Cécile Grohs, Carole Iampietro. DNA isolation from cattle semen for long read sequencing v1. 2024, 10.17504/protocols.io.j8nlkw1qwl5r/v1. hal-04414012

HAL Id: hal-04414012

https://hal.inrae.fr/hal-04414012v1

Submitted on 24 Jan 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_3.png" /></figure>

JAN 18, 2024

<figure><img src="image_4.png" /></figure>

# DNA isolation from cattle semen for long read sequencing

Erwan Denis¹, Cecile CG Grohs², Carole lampietro¹

¹INRAE, US 1426, GeT-PlaGe, Genotoul, France Genomique, Université Fédérale de Toulouse, Castanet-Tolosan, France;

²Université Paris-Saclay, INRAE, AgroParisTech, GABI, 78350, Jouy-en-Josas, France

<figure><img src="image_5.png" /><figcaption>Carole lampietro</figcaption></figure>

INRAE, GeT-PlaGe, Genotoul, France

## 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.

## 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.

## OPEN ACCESS

<figure><img src="image_6.png" /></figure>

### DOI:

dx.doi.org/10.17504/protocols.io.j8nlkw1qwl5r/v1

**Protocol Citation:** Erwan Denis, Cecile CG Grohs, Carole lampietro 2024. DNA isolation from cattle semen for long read sequencing. protocols.io

https://dx.doi.org/10.17504/protocols.io.j8nlkw1qwl5r/v1

**License:** This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

**Protocol status:** Working

We use this protocol and it's working

Created: Mar 24, 2023
<figure><img src="image_2.png" /></figure>

JAN 18, 2024

<figure><img src="image_3.png" /></figure>

# DNA isolation from cattle semen for long read sequencing

Erwan Denis¹, Cecile CG Grohs², Carole lampietro¹

¹INRAE, US 1426, GeT-PlaGe, Genotoul, France Genomique, Université Fédérale de Toulouse, Castanet-Tolosan, France;

²Université Paris-Saclay, INRAE, AgroParisTech, GABI, 78350, Jouy-en-Josas, France

<figure><img src="image_4.png" /><figcaption>Carole lampietro</figcaption></figure>

INRAE, GeT-PlaGe, Genotoul, France

## 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.

## 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.

## OPEN ACCESS

<figure><img src="image_5.png" /></figure>

### DOI:

dx.doi.org/10.17504/protocols.io.j8nlkw1qwl5r/v1

**Protocol Citation:** Erwan Denis, Cecile CG Grohs, Carole lampietro 2024. DNA isolation from cattle semen for long read sequencing. protocols.io

https://dx.doi.org/10.17504/protocols.io.j8nlkw1qwl5r/v1

**License:** This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

**Protocol status:** Working

We use this protocol and it's working

Created: Mar 24, 2023
**Last Modified**: Jan 18, 2024

**PROTOCOL integer ID**: 79397

**Keywords**: extraction, high molecular weight, sperm, DNA, Long read sequencing, bovine, DNA isolation, PacBio

**Funders Acknowledgement**: European Union and Occitanie region

Grant ID: Operational Program FEDER-FSE MIDI-PYRENEES ET GARONNE 2014-2020

## PROTOCOL MATERIALS

<figure><img src="image_7.png" /></figure>

Puregene Tissue Kit Qiagen Catalog #158063

Step 5

<figure><img src="image_8.png" /></figure>

Proteinase K Qiagen Catalog #19133

Step 5

<figure><img src="image_9.png" /></figure>

Tris(2-carboxyethyl)phosphine hydrochloride solution Merck MilliporeSigma (Sigma-Aldrich) Catalog #646547-10X1ML

Step 1

<figure><img src="image_10.png" /></figure>

Buffer RLT Qiagen Catalog #79216

Step 1

<figure><img src="image_11.png" /></figure>

Phosphate-buffered saline, pH 7.4

Step 2

<figure><img src="image_12.png" /></figure>

DNA LoBind Tubes 2.0 mL Eppendorf Catalog #30108078

Step 2

<figure><img src="image_13.png" /></figure>

EB buffer Qiagen Catalog #19086

Step 9

<figure><img src="image_14.png" /></figure>

Isopropanol

Step 8

## SAFETY WARNINGS

<figure><img src="image_15.png" /></figure>

See Safety Data Sheets for warnings and safety hazards.

## BEFORE START INSTRUCTIONS

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.

## Preparation of reagents

1. Immediately 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 follows:

<figure><img src="image_16.png" /></figure>

450 µL RTL

<figure><img src="image_17.png" /></figure>

50 µL TCEP

<figure><img src="image_18.png" /></figure>

Buffer RLT Qiagen Catalog #79216

<figure><img src="image_19.png" /></figure>

Tris(2-carboxyethyl)phosphine hydrochloride solution Merck MilliporeSigma (Sigma-Aldrich) Catalog #646547-10X1ML
**Last Modified:** Jan 18, 2024

**PROTOCOL integer ID:** 79397

**Keywords:** extraction, high molecular weight, sperm, DNA, Long read sequencing, bovine, DNA isolation, PacBio

**Funders Acknowledgement:**
European Union and Occitanie region

Grant ID: Operational Program FEDER-FSE MIDI-PYRENEES ET GARONNE 2014-2020

## PROTOCOL MATERIALS

Puregene Tissue Kit Qiagen Catalog #158063

Step 5

Proteinase K Qiagen Catalog #19133

Step 5

Tris(2-carboxyethyl)phosphine hydrochloride solution Merck MilliporeSigma (Sigma-Aldrich) Catalog #646547-10X1ML

Step 1

Buffer RLT Qiagen Catalog #79216

Step 1

Phosphate-buffered saline, pH 7.4

Step 2

DNA LoBind Tubes 2.0 mL Eppendorf Catalog #30108078

Step 2

EB buffer Qiagen Catalog #19086

Step 9

Isopropanol

Step 8

## SAFETY WARNINGS

<figure><img src="image_6.png" /></figure>

See Safety Data Sheets for warnings and safety hazards.

## BEFORE START INSTRUCTIONS

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.

## Preparation of reagents

1. Immediately 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:

<figure><img src="image_7.png" /></figure>

450 µL RTL

<figure><img src="image_8.png" /></figure>

50 µL TCEP

<figure><img src="image_9.png" /></figure>

Buffer RLT Qiagen Catalog #79216

<figure><img src="image_10.png" /></figure>

Tris(2-carboxyethyl)phosphine hydrochloride solution Merck MilliporeSigma (Sigma-Aldrich) Catalog #646547-10X1ML
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.

LINK

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..

LINK

https://doi.org/10.2144/000114280

Preparation of sample

2 **Recovery of spermatozoa from the straw:**

- Empty the

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

Sample

in a

<figure><img src="image_22.png" /></figure>

2 mL tube by cutting the two ends of the straw

<figure><img src="image_23.png" /></figure>

DNA LoBind Tubes 2.0 mL Eppendorf Catalog #30108078

- Rince the straw it with

<figure><img src="image_24.png" /></figure>

200 µL 1X PBS

<figure><img src="image_25.png" /></figure>

Room temperature

<figure><img src="image_26.png" /></figure>

Phosphate-buffered saline, pH 7.4 **Contributed by users**

3 **Wash:**

10m

- Add

<figure><img src="image_27.png" /></figure>

800 µL

more PBS (up to

<figure><img src="image_28.png" /></figure>

1 mL 1X PBS

)

- Pellet

<figure><img src="image_29.png" /></figure>

1000 x g, Room temperature, 00:05:00

- Discard the supernatant

Second wash is optional (no significant impact observed)

-Re-suspend in

<figure><img src="image_30.png" /></figure>

1 mL 1X PBS

-Pellet

<figure><img src="image_31.png" /></figure>

1000 x g, Room temperature, 00:05:00

-Discard the supernatant
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.

LINK

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..

LINK

https://doi.org/10.2144/000114280

Preparation of sample

2 **Recovery of spermatozoa from the straw:**

- Empty the

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

Sample

in a

<figure><img src="image_13.png" /></figure>

2 mL tube

by cutting the two ends of the straw

<figure><img src="image_14.png" /></figure>

DNA LoBind Tubes 2.0 mL Eppendorf Catalog #30108078

- Rince the straw it with

<figure><img src="image_15.png" /></figure>

200 µL 1X PBS

<figure><img src="image_16.png" /></figure>

Room temperature

<figure><img src="image_17.png" /></figure>

Phosphate-buffered saline, pH 7.4 **Contributed by users**

3 **Wash:**

10m

- Add

<figure><img src="image_18.png" /></figure>

800 µL

more PBS (up to

<figure><img src="image_19.png" /></figure>

1 mL 1X PBS

)

- Pellet

<figure><img src="image_20.png" /></figure>

1000 x g, Room temperature, 00:05:00

- Discard the supernatant

Second wash is optional (no significant impact observed)

-Re-suspend in

<figure><img src="image_21.png" /></figure>

1 mL 1X PBS

-Pellet

<figure><img src="image_22.png" /></figure>

1000 x g, Room temperature, 00:05:00

-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:** 10m 10s

Add Ⓐ 500 µL of RLT-TCEP to the pellet

- Vortex ⏻ 00:00:10 by pulsing at max speed

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

- Incubate °C On ice ℹ 00:10:00

5 **Step two:** continue with Qiagen Puregene Tissue kit adapted as follow 1h 30m

- Add Ⓐ 500 µL of Cell Lysis Solution

- Add Ⓐ 60 µL of [M] 20 mg/mL proteinase K (20 mg/ml)

- Mix by inversion (about 25 inversions)

- Incubate °C 55 °C ℹ 01:30:00

<figure><img src="image_32.png" /></figure>

Puregene Tissue Kit Qiagen Catalog #158063

<figure><img src="image_33.png" /></figure>

Proteinase K Qiagen Catalog #19133

6 **Remove RNA:** 16m

- Ⓐ 3 µL RNAse from Qiagen Puregene Tissue Kit

- Incubate °C 37 °C ℹ 00:15:00

- Incubate °C On ice ℮ 00:01:00

Protein precipitation

1m 15s

7 - Add Ⓐ 200 µL of Protein precipitation buffer (from Qiagen Puregene Tissue Kit) 6m 15s

- Mix by hand or gently vortexing ℮ 00:00:15

- Incubate °C On ice ℮ 00:05:00

- Centrifuge ⚙️ 16000 x g, Room temperature, 00:01:00

DNA precipitation

6m

8 - Transfer the supernatant to a new tube containing Ⓐ 600 µL of Isopropanol 6m

- Carefully invert the tube 25-50X times to form the pellet

- Incubate ℮ 00:05:00 °C Room temperature

<figure><img src="image_34.png" /></figure>

<figure><img src="image_35.png" /></figure>
Note

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

Lysis

4

**Step one:**

10m 10s

Add ⬇ 500 µL of RLT-TCEP to the pellet

- Vortex ⏻ 00:00:10 by pulsing at max speed

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

- Incubate ⌒ On ice ⏻ 00:10:00

5

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

1h 30m

- Add ⬇ 500 µL of Cell Lysis Solution

- Add ⬇ 60 µL of [M] 20 mg/mL proteinase K (20 mg/ml)

- Mix by inversion (about 25 inversions)

- Incubate ⌒ 55 °C ⏻ 01:30:00

<figure><img src="image_23.png" /></figure>

Puregene Tissue Kit Qiagen Catalog #158063

<figure><img src="image_24.png" /></figure>

Proteinase K Qiagen Catalog #19133

6

**Remove RNA:**

16m

- ⬇ 3 µL RNAse from Qiagen Puregene Tissue Kit

- Incubate ⌒ 37 °C ⏻ 00:15:00

- Incubate ⌒ On ice ⏻ 00:01:00

Protein precipitation

1m 15s

7

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

6m 15s

- Mix by hand or gently vortexing ⏻ 00:00:15

- Incubate ⌒ On ice ⏻ 00:05:00

- Centrifuge ⚙️ 16000 x g, Room temperature, 00:01:00

DNA precipitation

6m

8

- Transfer the supernatant to a new tube containing ⬇ 600 µL of Isopropanol

6m

- Carefully invert the tube 25-50X times to form the pellet

- Incubate ⌒ 00:05:00 ⌅ Room temperature

<figure><img src="image_25.png" /></figure>

<figure><img src="image_26.png" /></figure>
- Centrifuge ⏸ 16000 x g, 00:01:00

- Discard supernatant

<figure><img src="image_36.png" /></figure>

Isopropanol **Contributed by users**

9

- Add ⏻ 600 µL of 70% ethanol to the pellet

<figure><img src="image_37.png" /></figure>

- Centrifuge ⏸ 5000 x g, 00:02:00

<figure><img src="image_38.png" /></figure>

- Discard supernatant

- Almost dry the pellet 🔧 Room temperature 🕒 00:05:00

<figure><img src="image_39.png" /></figure>

<figure><img src="image_40.png" /></figure>

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

<figure><img src="image_41.png" /></figure>

<figure><img src="image_42.png" /></figure>

- Store DNA at 4°C

<figure><img src="image_43.png" /></figure>

EB buffer **Qiagen Catalog #19086**

**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.

**Expected result**

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..

**LINK**

https://doi.org/10.1101/gr.277787.123
- Centrifuge ⏸ 16000 x g, 00:01:00

- Discard supernatant

<figure><img src="image_27.png" /></figure>

Isopropanol **Contributed by users**

9

- Add ⏻ 600 µL of 70% ethanol to the pellet

<figure><img src="image_28.png" /></figure>

- Centrifuge ⏸ 5000 x g, 00:02:00

<figure><img src="image_29.png" /></figure>

- Discard supernatant

- Almost dry the pellet 🔧 Room temperature 🕒 00:05:00

<figure><img src="image_30.png" /></figure>

<figure><img src="image_31.png" /></figure>

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

<figure><img src="image_32.png" /></figure>

<figure><img src="image_33.png" /></figure>

- Store DNA at 4°C

<figure><img src="image_34.png" /></figure>

EB buffer Qiagen Catalog #19086

**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.

**Expected result**

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..

**LINK**

https://doi.org/10.1101/gr.277787.123