|
| Status |
Public on Jun 09, 2009 |
| Title |
H4K12ac ChIP, biological replicate 1, chip1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
DNA associated with histone H4 acetylated at lysine 12 (H4K12ac)
|
| Organism |
Homo sapiens |
| Characteristics |
antibody: H4K12ac
cell type: spermatozoa
|
| Treatment protocol |
Chromatin immunoprecipitation (ChIP) was performed using ChIP assay kit (Cat¹17-295) provided by UpstateMillipore (http://www.millipore.com) with modifications due to the relatively low quantity of histones in spermatozoa
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Approximately 107 cells from fertile donors were separated from seminal plasma and washed twice in 1x Dulbecco’s PBS by 5 min centrifugation at 300 x g. Chromatin crosslinking was carried out by addition of formaldehyde to a final volume of 1% and 10 min incubation on a rotating platform at room temperature followed by 10 min incubation with 125 mM glycine to quench the crosslinking reaction. After fixation, sperm was pelleted by 10 min centrifugation at 300 x g and resuspended in 200 µl cold PBS containing protease inhibitors (Roche, Mannheim, Germany). To separate heads from tails, cells were homogenised with 20 strokes in an UltraTurrax homogeniser (IKA, Staufen, Germany). After adding 2 ml lysis buffer, the sample was sonicated 10 times on ice with a Branson 250 sonifier on setting 3, duty cycle 50 % for 30 sec to obtain an average fragment length of 200-1000 bp. Chromatin was then diluted 10-fold with the provided dilution buffer. 1 ml of the probe was precleared with 40 µl of salmon sperm DNA/protein A agarose solution before overnight incubation at 4°C with primary antibodies purchased from Abcam (Cambridge, UK). These included a polyclonal antibody recognising histone H4 acetylated at lysine 12 (H4K12ac) (ab1761); a non-specific rabbit polyclonal IgG (ab46540) was used as a negative control and a polyclonal recognising unmodified histone H3 (abcam ab1791) was used as a positive control. 10 % of sonicated chromatin (100 µl) was retained from each sample to determine the amount of input DNA. After addition of 40 µl salmon sperm DNA/protein A agarose solution, samples were incubated for 2 hrs. Beads were then washed with the provided buffers and the chromatin was eluted twice with 250 µl elution buffer (1 % SDS in 0.1 M NaHCO3). Crosslinks were reversed by addition of 5 M NaCl and incubation at 65 °C for 4 hrs followed by proteinase K digestion at 45 °C for 1 hr. DNA was extracted by phenol/chloroform using ethanol precipitation with 3 µl glycogen as inert carrier, resuspended in 30 µl nuclease free water and stored at –20 °C.
|
| Label |
Cy5
|
| Label protocol |
Labeling and hybridisation of the probes for ChIP-on-chip analysis was performed by NimbleGen Service ImaGenes GmbH (Berlin, Germany)
|
| |
|
| Channel 2 |
| Source name |
Total Unfractionated Chromatin
|
| Organism |
Homo sapiens |
| Characteristics |
antibody: n/a
cell type: spermatozoa
|
| Treatment protocol |
Chromatin immunoprecipitation (ChIP) was performed using ChIP assay kit (Cat¹17-295) provided by UpstateMillipore (http://www.millipore.com) with modifications due to the relatively low quantity of histones in spermatozoa.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Approximately 107 cells from fertile donors were separated from seminal plasma and washed twice in 1x Dulbecco’s PBS by 5 min centrifugation at 300 x g. Chromatin crosslinking was carried out by addition of formaldehyde to a final volume of 1% and 10 min incubation on a rotating platform at room temperature followed by 10 min incubation with 125 mM glycine to quench the crosslinking reaction. After fixation, sperm was pelleted by 10 min centrifugation at 300 x g and resuspended in 200 µl cold PBS containing protease inhibitors (Roche, Mannheim, Germany). To separate heads from tails, cells were homogenised with 20 strokes in an UltraTurrax homogeniser (IKA, Staufen, Germany). After adding 2 ml lysis buffer, the sample was sonicated 10 times on ice with a Branson 250 sonifier on setting 3, duty cycle 50 % for 30 sec to obtain an average fragment length of 200-1000 bp. Chromatin was then diluted 10-fold with the provided dilution buffer. 1 ml of the probe was precleared with 40 µl of salmon sperm DNA/protein A agarose solution before overnight incubation at 4°C with primary antibodies purchased from Abcam (Cambridge, UK). These included a polyclonal antibody recognising histone H4 acetylated at lysine 12 (H4K12ac) (ab1761); a non-specific rabbit polyclonal IgG (ab46540) was used as a negative control and a polyclonal recognising unmodified histone H3 (abcam ab1791) was used as a positive control. 10 % of sonicated chromatin (100 µl) was retained from each sample to determine the amount of input DNA. After addition of 40 µl salmon sperm DNA/protein A agarose solution, samples were incubated for 2 hrs. Beads were then washed with the provided buffers and the chromatin was eluted twice with 250 µl elution buffer (1 % SDS in 0.1 M NaHCO3). Crosslinks were reversed by addition of 5 M NaCl and incubation at 65 °C for 4 hrs followed by proteinase K digestion at 45 °C for 1 hr. DNA was extracted by phenol/chloroform using ethanol precipitation with 3 µl glycogen as inert carrier, resuspended in 30 µl nuclease free water and stored at –20 °C.
|
| Label |
Cy3
|
| Label protocol |
Labeling and hybridisation of the probes for ChIP-on-chip analysis was performed by NimbleGen Service ImaGenes GmbH (Berlin, Germany)
|
| |
|
| |
| Hybridization protocol |
Hybridization was conducted at the manufacturer's facility
|
| Scan protocol |
Scanning was conducted at the manufacturer's facility
|
| Description |
For chip array, purified IP-probe (Qiaquick Purification Kit, Qiagen, Hilden, Germany) and 10 ng of input material (total chromatin) was prepared by adapting the protocol for whole genome amplification using Sigma GenomePlex WGA kit (http://www.sigmaldrich.com) as described in O’Green et al. {O'Geen, 2006 #170}. The initial random amplification step of the WGA protocol was omitted due to previous defragmentation of sperm chromatin after sonification. The required amount of DNA for microarray analysis (4 µg per sample) was generated using WGA Reamplifikation Kit (Sigma, Munich, Germany). Amplicons were applied to the HG18 human 5 kb promoter array from NimbleGen System (http://www.nimblegen.com).
|
| Data processing |
Fluorescence signal intensity data were extracted from the scanned images of each array using Nimblegen data extraction software. Each feature on the array has a corresponding scaled log2-ratio that was calculated from the input signal (Cy3 for total chromatin) and IP-probe (Cy5) which were co-hybridized to the array. The log2 ratio was computed and scaled to center the ratio data around zero.
|
| |
|
| Submission date |
May 21, 2009 |
| 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 |
GPL6325 |
| Series (1) |
| GSE13101 |
H4K12 acetylation in Human Spermatozoa |
|
