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# Goal/Experiment:
The goal of this experiment is to isolate human sperm DNA, treating it with bisulfite, and performing pyrosequencing to analyze the methylation status of specific DNA regions.

# Bisulfite Pyrosequencing Protocol for Human Sperm DNA
**Version 2**

**Zhaoxu Lu, Mei Qiang**

**Published: 30 Mar 2018**

## Abstract
Citation: Zhaoxu Lu, Mei Qiang Bisulfite pyrosequencing protocol for Human sperm DNA. protocols.io dx.doi.org/10.17504/protocols.io.ns2dg8e

## Protocol

### Human Sperm DNA Isolation Procedure

#### Step 1
**Extraction buffer of sperm DNA**

1. 21.2 ml 6mol/L guanidine thiocyanate
2. 600 µl 5mol/L NaCl
3. 1 ml 30% N-lauroylsarcosine sodium salt
4. 3 ml 1mol/l dithiothreitol (DTT)
5. 600 µl 10mg/ml proteinase K
6. 3.6 ml Doubly deionized water

#### Step 2
Add 150 µl of semen to a 1.5 ml micro-centrifuge tube.

#### Step 3
Wash with 1 ml of PBS (0.1 mol/L).

#### Step 4
Centrifuge at 1500 ×g for 10 min at 4°C.

#### Step 5
Repeat washing 2 times as described in step 3-4.

#### Step 6
Add 0.5 ml extraction buffer into sperm pellet.

#### Step 7
Place in a 65°C water bath for 12 hours.

#### Step 8
Cool at room temperature.

#### Step 9
Add 10 µl of RNase A (10 mg/ml), mix by pulse-vortexing for 15 seconds, and incubate for 10 min at room temperature.

#### Step 10
Briefly centrifuge the tube.

#### Step 11
Add 510 µl of isopropanol and centrifuge at 10000 ×g for 10 min at 4°C.

#### Step 12
Add 800 µl of ethanol (75%), and reverse mix for multiple times.

#### Step 13
Incubate for 12 hours at -20°C.

#### Step 14
Centrifuge at 10000 ×g for 10 min at 4°C. Then dry sample at room temperature.

#### Step 15
Sperm DNA is dissolved in 50 µl of Elution Buffer.

#### Step 16
Incubate in a 65°C water bath for 2 hours.

### Procedure for Bisulfite Treatment

#### Step 17
Add 130 µl of the CT Conversion Reagent solution to 1000 ng of your DNA sample in a PCR tube.

#### Step 18
Place the sample tube in a thermal cycler and perform the following steps:
- 98°C for 10 minutes
- 64°C for 2.5 hours
- 4°C

#### Step 19
Add 600 µl of M-Binding Buffer into a Zymo-Spin IC™ Column and place the column into a provided Collection Tube.

#### Step 20
Load sample (from Step 2) into the Zymo-Spin IC™ Column containing the M-Binding Buffer. Close the cap and mix by inverting the column several times.

#### Step 21
Centrifuge at full speed (>10,000 ×g) for 30 seconds. Discard the flow-through.

#### Step 22
Add 100 µl of M-Wash Buffer to the column. Spin at full speed for 30 seconds.

#### Step 23
Add 200 µl of M-Desulphonation Buffer to the column and let stand at room temperature (20°C-30°C) for 15-20 minutes. After the incubation, spin at full speed for 30 seconds.

#### Step 24
Add 200 µl of M-Wash Buffer to the column. Spin at full speed for 30 seconds. Add another 200 µl of M-Wash Buffer and spin at top speed for 30 seconds.

#### Step 25
Add 8 µl of M-Elution Buffer directly to the column matrix. Place the column into a 1.5 ml tube. Spin briefly at full speed to elute the DNA. Add 7 µl of M-Elution Buffer and additional repeated 1-time eluting was subsequently performed.

#### Step 26
The DNA is ready for immediate analysis or can be stored at or below -20°C for later use.

### PCR Amplification of Bisulfite-Treated Sperm DNAs

#### Step 27
All reactions are performed with provided PCR mixtures (total volume at 25 µl) provided in Table 1. Each reaction also contains 2.5 µl of CoralLoad Concentrate (10x) for checking amplicons on an agarose gel.

| Components | Volume (µl) | Final concentration |
|------------|-------------|---------------------|
| PyroMark PCR Master Mix, 2x | 12.5 | 1x |
| CoraLoad Concentrate, 10x | 2.5 | 1x |
| Q-solution, 5x | 5 | 1x |
| Primer forward (10 uM) | 0.5 | 0.2uM |
| Primer reverse (10 uM) | 0.5 | 0.2uM |
| Template DNA | 50ng | |
| Final volume | 25 | |

#### Step 28
PCR and pyrosequencing primers are designed and listed in Table 2. Reverse primer is conjugated to biotin.

| Primer | Sequence |
|--------|----------|
| DMR Forward primer | * |
| Reverse primer | * |
| Sequencing primer H19 | GTATATGGGTATTTTTTGAGGTT |
| H19 | ATAATCCGTATTCCAAAATA |
| Sequence primer | * |
| DMR-PCR product | * |
| Sequence primer | * |
| H19 | * |
| Sequencing primer H19 | * |
| H19 | * |

#### Step 29
The PCR conditions are as following:
- 94 °C for 15 minutes
- 45 cycles of:
  - 94 °C, 30 secs
  - 56 °C, 30 secs
  - 72 °C, 30 secs
- Final extension step:
  - 72 °C for 10 minutes

### Pyrosequencing

#### Step 30
Add 40 µl of Binding Buffer, 3 µl of streptavidin-sepharose beads, and 17 µl DDW into 20 µl of PCR products.

#### Step 31
Seal film and shake at 1400 rpm for 10 min to room temperature.

#### Step 32
PCR products on streptavidin-sepharose beads are washed with ethanol (10%) for 5 seconds.

#### Step 33
Place sample (from Step 31) into denaturation solution for 5 seconds.

#### Step 34
Place sample (from Step 31) into Wash Buffer for 10 seconds for getting purified biotinylated single stranded PCR products. These single stranded PCR products are isolated using the Pyrosequencing Work Station.

#### Step 35
Transfer purified biotinylated single stranded PCR products into PSQ 96 Plate Low with 40 µl annealing buffer and 1.6 µl sequencing primer (10µmol/L).

#### Step 36
Heat PSQ 96 Plate Low at 80 °C for 2 minutes.

#### Step 37
Undergo pyrosequencing on a Pyromark Q96 MD pyrosequencing instrument and sequence using PyroMark Gold Q96 kit.

#### Step 38
The degree of methylation at each CpG site is determined using PyroMark CpG Software (Biotage AB, Uppsala, Sweden). Pyrosequencing assays are performed in duplicate in sequential runs (technical replicates), and the values show represent the mean methylation for each individual CpG site.

**endofoutput**
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