|
| Status |
Public on Jun 09, 2009 |
| Title |
DNA packaging in human spermatozoal chromatin:SRDI versus SRDS (44K3)-dye swap |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Spermatozoal DNA partitioned into a Salt Insoluble (SRDI) Fraction
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Spermatozoa
donor: healthy human donor
|
| Treatment protocol |
Semen was washed free of seminal plasma by diluting the semen 1:1 with HEPES buffered HSOF and centrifuging for 10 minutes at 500 x g. Pellets were resuspended in HSOF and pelleted as before. This was repeated until the supernatants were clear. Pellets were resuspended in STE buffer (100mM NaCl, 10 mM Tris-Cl, pH 8.0, 1 mM EDTA) containing 0.2 mM phenylmethylsulphonylfluoride (PMSF) and re-pelleted to remove HSOF. Pellets were subsequently processed with sequential extraction in 10% (w/v) mixed CTAB (alkly-trimethylammonium bromide), which removes the flagella, including principal pieces, thus ensuring no contamination of the samples with non-genomic DNA followed by sequential incubation in 1% w/v CTAB in 10 mM Tris-Cl pH 8.0 containing 0.05% w/v digitonin; Tris buffered saline (TBS) containing 0.05% w/v digitonin, 10 mM Tris-Cl pH 8.0 containing 1mM EDTA, 0.05% w/v digitonin, EDTA-free protease inhibitor cocktail (Roche) and finally extraction in 0.65M NaCl to release soluble histones. Nuclear pellets were re-suspended in BamH1 buffer containing 0.05% digitonin and digested with ~150 units each of BamH1 and EcoR1 (Invitrogen, Paisley, UK: 2,000 U per 5 x 108 nuclei) for 90 minutes at 37°C. Suspensions were centrifuged at 3,000 x g for 5 minutes and the supernatants removed. STOP buffer comprising proteinase K (Bioline: - 200 µg/ml) in TBS, 0.5% w/v sodium dodecyl sulphate (SDS) was added to this suspension and incubated for 10 hours at 55°C. Pellets were also resuspended in STOP buffer and concurrently digested under identical conditions. DNA was isolated from the insoluble fraction by incubation in STOP buffer containing RNAse A at 10mg/ml for 1 hour at 37°C and extraction in phenol chloroform (1:1) followed by ethanol precipitation. Precipitated DNA was dissolved in TE buffer and stored at 4°C.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
CGH profiling was carried out according to the manufacturer's (Agilent) instructions. Prior to processing, all DNA samples (including those pre-digested with EcoR1 and BamH1 or MNase were double re-digested with 10 units each of Alu1 and Rsa1 (New England Biolabs, Hitchin, UK) that generates fragments of 500 bp or less.
|
| Label |
Cy5
|
| Label protocol |
Labelling with cy3 or cy5 (GE Healthcare, Amersham UK) was accomplished using a random priming method in the presence of Klenow DNA polymerase (Invitrogen, Paisley, UK) according to the suggested Agilent protocol. Unincorporated nucleotides were removed by passing the reaction mixture through a sephadex G50 column.
|
| |
|
| Channel 2 |
| Source name |
Spermatozoal DNA partitioned into a Salt Soluble (SRDS) Fraction
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Spermatozoa
donor: healthy human donor
|
| Treatment protocol |
Semen was washed free of seminal plasma by diluting the semen 1:1 with HEPES buffered HSOF and centrifuging for 10 minutes at 500 x g. Pellets were resuspended in HSOF and pelleted as before. This was repeated until the supernatants were clear. Pellets were resuspended in STE buffer (100mM NaCl, 10 mM Tris-Cl, pH 8.0, 1 mM EDTA) containing 0.2 mM phenylmethylsulphonylfluoride (PMSF) and re-pelleted to remove HSOF. Pellets were subsequently processed with sequential extraction in 10% (w/v) mixed CTAB (alkly-trimethylammonium bromide), which removes the flagella, including principal pieces, thus ensuring no contamination of the samples with non-genomic DNA followed by sequential incubation in 1% w/v CTAB in 10 mM Tris-Cl pH 8.0 containing 0.05% w/v digitonin; Tris buffered saline (TBS) containing 0.05% w/v digitonin, 10 mM Tris-Cl pH 8.0 containing 1mM EDTA, 0.05% w/v digitonin, EDTA-free protease inhibitor cocktail (Roche) and finally extraction in 0.65M NaCl to release soluble histones.The supernatant following salt extraction was retained. DNA was isolated from the soluble fraction by incubation in STOP buffer containing RNAse A at 10mg/ml for 1 hour at 37°C and extraction in phenol chloroform (1:1) followed by ethanol precipitation. Precipitated DNA was dissolved in TE buffer and stored at 4°C.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
CGH profiling was carried out according to the manufacturer's (Agilent) instructions. Prior to processing, all DNA samples (including those pre-digested with EcoR1 and BamH1 or MNase were double re-digested with 10 units each of Alu1 and Rsa1 (New England Biolabs, Hitchin, UK) that generates fragments of 500 bp or less.
|
| Label |
Cy3
|
| Label protocol |
Labelling with cy3 or cy5 (GE Healthcare, Amersham UK) was accomplished using a random priming method in the presence of Klenow DNA polymerase (Invitrogen, Paisley, UK) according to the suggested Agilent protocol. Unincorporated nucleotides were removed by passing the reaction mixture through a sephadex G50 column.
|
| |
|
| |
| Hybridization protocol |
For hybridisation, equal quantities of labelled DNA (500 - 1000 ng) were mixed with Cot-1 DNA (1 mg/ml) and incubated at 95°C for 3 minutes and then re-incubated at 37°C for 30 minutes. The DNAs were applied to Agilent's 44K (100 µl) arrays, sealed and then incubated for 24 hours at 65°C with constant rotation (20 rpm).
|
| Scan protocol |
Scanning was conducted an accordance with the Agilent protocol.
|
| Description |
Spermatozoal DNA from a healthy human donor was partitioned through a salt extraction to profile protamine associated DNA relative to histone associated DNA.
|
| Data processing |
Data was processed for feature extraction, background subtraction and normalized in the Agilent CGH analytics software package.
The default settings for the Agilent Analytics CGH v9.1 protocols were used (selection of a subset of rank invariant probes and using these for a linear median normalization between channels).
|
| |
|
| Submission date |
Jul 12, 2008 |
| Last update date |
Jun 09, 2009 |
| Contact name |
David Miller |
| Organization name |
Leeds University
|
| Department |
Leeds Institute for Genetics and Health Therapeutics
|
| Lab |
Reproductive and Early Development Unit
|
| Street address |
Clarendon Way
|
| City |
Leeds |
| ZIP/Postal code |
LS2 9JT |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL2879 |
| Series (2) |
| GSE12088 |
DNA packaging of human spermatozoal chromatin |
| GSE13238 |
DNA packaging of human and murine spermatozoal chromatin |
|
