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| Status |
Public on Sep 30, 2015 |
| Title |
H3K9me3_swap |
| Sample type |
genomic |
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| Channel 1 |
| Source name |
H3K9me3 input DNA
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| Organism |
Drosophila melanogaster |
| Characteristics |
cell type: mature spermatozoa
chip antibody: none, input DNA
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For each ChIP replicate, 10E6 frozen spermatozoa were thawed at room temperature, washed in cold PBS and fixed in 100 μl 1% formaldehyde in PBS at room temperature for 10 minutes. Crosslinking was quenched by the addition of 400 μl 0.125 M glycine in PBS and incubation at 4C for 5 minutes. Cells were washed twice in 500 µl ice-cold PBS and incubated for 1 hour at room temperature in 500 µl lysis buffer A (10mM DTT, 10 mM HEPES pH8, 10 mM EDTA, 0.5 mM EGTA, 0.25% Triton X-100, 10mM DTT, 0.5% (w/v) SDS and protease inhibitors (10 mg/ml aprotinin, 5 mg/ml leupeptin, and 0.5 mM PMSF)). The DTT was quenched by the addition of N-ethylmaleimide to a final concentration of 5 mM and the cells were resuspended in 100 µl lysis buffer B (10 mM HEPES pH8, 200 mM NaCl, 0.5 mM EGTA, 1 mM EDTA, 0.01% Triton X-100 and protease inhibitors), pelleted and resuspended in 100 μl IP buffer (25 mM Tris pH8, 150 mM NaCl, 2 mM EDTA, 1%Triton X-100 and 7.5% glycerol) with 0.25% SDS and protease inhibitors. Chromatin was sheared to approximately 500 bp fragments by sonication on ice using a MEM Soniprep 150 sonicator at 6 micron amplitude for 5 cycles of 20 seconds sonication and 40 seconds cooling. Lysates were diluted in four volumes IP buffer and cleared by centrifugation. For each IP, a 10 μl suspension of Protein G Dynabeads (pre-absorbed with BSA and herring sperm DNA) was incubated in 500 μl 100 mM sodium phosphate pH8, 0.5% BSA containing 5 µg ChIP-grade antibody (or 5 µg goat anti-rabbit IgG antibody for mock IPs) at 4C with rotation for 2 hours. The Dynabeads were washed in IP buffer and resuspended in 490 μl cleared lysate along with 25 μg sonicated herring sperm DNA. Reactions were incubated at 4C with rotation for 4 hours. Immune complexes were recovered, suspended in 500 μl buffer 1 (20 mM Tris-HCl pH8, and 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS and protease inhibitors) and incubated on ice for 10 minutes before being washed twice in 500 μl buffer 2 (20 mM Tris-HCl pH8, 500 mM NaCl, 2mM EDTA, 1% Triton X-100, 0.1% SDS and protease inhibitors) for 10 minutes at 4C with rotation and once in 500 μl buffer 3 (10 mM Tris-HCl pH8, 0.25 M LiCl, 1% sodium deoxycholate, 1 mM EDTA, and 1% NP40) for 10 minutes at 4C with rotation. Finally, the beads were washed twice in ice-cold TNE buffer (10 mM Tris pH8, 1 mM EDTA, 50 mM NaCl). Chromatin was eluted from the beads in 50 μl 1% SDS, 0.1 M NaHCO3 pH10.1 at room temperature with rotation for 15 minutes. Eluates were transferred to fresh non-stick tubes, the procedure was repeated and the eluates were pooled. To each pooled eluate, 4 μl 5 M NaCl and 5 µl 0.2 M EDTA were added and eluates were incubated overnight at 55C. The pH of the eluates was adjusted to 8.3 by the addition of 4 μl 2 M Tris pH6.5. Residual RNAs were removed by the addition of 1 µl 10 mg/ml RNase A followed by incubation at 37C for 30 minutes, and proteins were removed by the addition of 1 μl 10 mg/ml proteinase K and incubation at 55C for 4 hours. Input DNA was extracted from the sonicated chromatin of 2 x 10E6 cells in 200 μl IP buffer by the addition of 10 µl 3 M sodium acetate pH7 and 10 µl 10 % SDS, followed by overnight incubation at 55C. Residual RNAs and proteins were removed as before. DNAs were recovered using a GeneluteTM PCR Clean-Up kit (Sigma-Aldrich) and quantified using a PicoGreen dsDNA Quant-iT(TM) kit (Invitrogen) according to manufacturers’ instructions. DNAs of input, IP and mock samples were amplified in two steps; firstly, DNA was amplified using a GenomePlex Single Cell kit (WGA4; Sigma-Aldrich), and secondly, the DNA was re-amplified using a GenomePlex Re-amplification Kit (WGA3; Sigma-Aldrich) according to the manufacturer’s instructions.
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| Label |
Cy5
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| Label protocol |
Random primers were annealed to 1 μg denatured DNA templates and extended by Klenow fragment incorporating Cy3- or Cy5-dCTP at the Flychip Microarray Facility (http://www.flychip.org.uk/protocols/chip/NimblegenBioPrime.php) using a Bioprime DNA Labeling System (Invitrogen; Cat. No. 18094-011).
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| Channel 2 |
| Source name |
H3K9me3 ChIP DNA
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| Organism |
Drosophila melanogaster |
| Characteristics |
cell type: mature spermatozoa
chip antibody: rabbit polyclonal anti-histone H3K9me3; Abcam ab8898
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For each ChIP replicate, 10E6 frozen spermatozoa were thawed at room temperature, washed in cold PBS and fixed in 100 μl 1% formaldehyde in PBS at room temperature for 10 minutes. Crosslinking was quenched by the addition of 400 μl 0.125 M glycine in PBS and incubation at 4C for 5 minutes. Cells were washed twice in 500 µl ice-cold PBS and incubated for 1 hour at room temperature in 500 µl lysis buffer A (10mM DTT, 10 mM HEPES pH8, 10 mM EDTA, 0.5 mM EGTA, 0.25% Triton X-100, 10mM DTT, 0.5% (w/v) SDS and protease inhibitors (10 mg/ml aprotinin, 5 mg/ml leupeptin, and 0.5 mM PMSF)). The DTT was quenched by the addition of N-ethylmaleimide to a final concentration of 5 mM and the cells were resuspended in 100 µl lysis buffer B (10 mM HEPES pH8, 200 mM NaCl, 0.5 mM EGTA, 1 mM EDTA, 0.01% Triton X-100 and protease inhibitors), pelleted and resuspended in 100 μl IP buffer (25 mM Tris pH8, 150 mM NaCl, 2 mM EDTA, 1%Triton X-100 and 7.5% glycerol) with 0.25% SDS and protease inhibitors. Chromatin was sheared to approximately 500 bp fragments by sonication on ice using a MEM Soniprep 150 sonicator at 6 micron amplitude for 5 cycles of 20 seconds sonication and 40 seconds cooling. Lysates were diluted in four volumes IP buffer and cleared by centrifugation. For each IP, a 10 μl suspension of Protein G Dynabeads (pre-absorbed with BSA and herring sperm DNA) was incubated in 500 μl 100 mM sodium phosphate pH8, 0.5% BSA containing 5 µg ChIP-grade antibody (or 5 µg goat anti-rabbit IgG antibody for mock IPs) at 4C with rotation for 2 hours. The Dynabeads were washed in IP buffer and resuspended in 490 μl cleared lysate along with 25 μg sonicated herring sperm DNA. Reactions were incubated at 4C with rotation for 4 hours. Immune complexes were recovered, suspended in 500 μl buffer 1 (20 mM Tris-HCl pH8, and 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS and protease inhibitors) and incubated on ice for 10 minutes before being washed twice in 500 μl buffer 2 (20 mM Tris-HCl pH8, 500 mM NaCl, 2mM EDTA, 1% Triton X-100, 0.1% SDS and protease inhibitors) for 10 minutes at 4C with rotation and once in 500 μl buffer 3 (10 mM Tris-HCl pH8, 0.25 M LiCl, 1% sodium deoxycholate, 1 mM EDTA, and 1% NP40) for 10 minutes at 4C with rotation. Finally, the beads were washed twice in ice-cold TNE buffer (10 mM Tris pH8, 1 mM EDTA, 50 mM NaCl). Chromatin was eluted from the beads in 50 μl 1% SDS, 0.1 M NaHCO3 pH10.1 at room temperature with rotation for 15 minutes. Eluates were transferred to fresh non-stick tubes, the procedure was repeated and the eluates were pooled. To each pooled eluate, 4 μl 5 M NaCl and 5 µl 0.2 M EDTA were added and eluates were incubated overnight at 55C. The pH of the eluates was adjusted to 8.3 by the addition of 4 μl 2 M Tris pH6.5. Residual RNAs were removed by the addition of 1 µl 10 mg/ml RNase A followed by incubation at 37C for 30 minutes, and proteins were removed by the addition of 1 μl 10 mg/ml proteinase K and incubation at 55C for 4 hours. Input DNA was extracted from the sonicated chromatin of 2 x 10E6 cells in 200 μl IP buffer by the addition of 10 µl 3 M sodium acetate pH7 and 10 µl 10 % SDS, followed by overnight incubation at 55C. Residual RNAs and proteins were removed as before. DNAs were recovered using a GeneluteTM PCR Clean-Up kit (Sigma-Aldrich) and quantified using a PicoGreen dsDNA Quant-iT(TM) kit (Invitrogen) according to manufacturers’ instructions. DNAs of input, IP and mock samples were amplified in two steps; firstly, DNA was amplified using a GenomePlex Single Cell kit (WGA4; Sigma-Aldrich), and secondly, the DNA was re-amplified using a GenomePlex Re-amplification Kit (WGA3; Sigma-Aldrich) according to the manufacturer’s instructions.
|
| Label |
Cy3
|
| Label protocol |
Random primers were annealed to 1 μg denatured DNA templates and extended by Klenow fragment incorporating Cy3- or Cy5-dCTP at the Flychip Microarray Facility (http://www.flychip.org.uk/protocols/chip/NimblegenBioPrime.php) using a Bioprime DNA Labeling System (Invitrogen; Cat. No. 18094-011).
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| Hybridization protocol |
Labelled DNAs were precipitated with 0.1 volume 5 M NaCl and 1 volume isopropanol. ChIP and input DNAs were quantified and pooled in 43 μl hybridization buffer (NimbleGen Hybridization Kit, LS; Roche-Nimlegen; Cat. No. 05583934001); 41 μl volumes were hybridized for 20 hours at 42C to Nimblegen 2.1M arrays in a NimbleGen Hybridization System 4. Slides were washed in Nimblegen Wash buffers I, II and III according to manufacturer's instructions.
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| Scan protocol |
Slides were scanned using a GenePix 4000B Microarray Scanner according to manufacturer's instructions.
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| Description |
ChIP-chip spermatozoal chromatin anti-H3K9me3
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| Data processing |
Data were collected and processed using NimbleScan software as outlined in the Nimblegen Arrays User's Guide described at http://www.flychip.org.uk/protocols/chip/NimblegenBioPrime.php.
scaled log2 ratios (Cy5/Cy3)
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| Submission date |
Nov 07, 2013 |
| Last update date |
Sep 30, 2015 |
| Contact name |
David Edwin Iles |
| E-mail(s) |
daveiles@omicsltd.com
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| Phone |
0044 1943864567
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| Organization name |
Omics Ltd.
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| Street address |
50 Hall Drive
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| City |
Burley-in-Wharfedale |
| State/province |
West Yorkshire |
| ZIP/Postal code |
LS29 7LP |
| Country |
United Kingdom |
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| Platform ID |
GPL15641 |
| Series (1) |
| GSE52165 |
Nucleosomal chromatin in the mature sperm of Drosophila melanogaster. |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM1260472_509062_H3K9_4_532.pair.gz |
33.0 Mb |
(ftp)(http) |
PAIR |
| GSM1260472_509062_H3K9_4_635.pair.gz |
33.8 Mb |
(ftp)(http) |
PAIR |
| GSM1260472_H3K9me3_swap_processed.txt.gz |
25.1 Mb |
(ftp)(http) |
TXT |
| Processed data provided as supplementary file |
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