|
| Status |
Public on Oct 13, 2011 |
| Title |
expt.1395 extraction4_array1 |
| Sample type |
genomic |
| |
|
| Source name |
expt.1395 extraction4_array1 channel_1
|
| Organism |
Drosophila melanogaster |
| Characteristics |
cell line: S2-DRSC
tissue: embryo-derived cell-line
developmental stage: late embryonic stage
Sex: Male
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The template for the dsRNA synthesis was PCR amplified from Drosophila genomic DNA using specific primers extended with 5? T7-promoter tails. The sequence of the T7-promoter tails is as follows: 5?- CTAATACGACTCACTATAGGGAG-3?. The amplicon was selected such that it is contained within single exon. Following PCR the template was purified using PCR purification Kit from Qiagen. 2.5ug of purified template was used to synthesize dsRNA with Ribomax T7 RNA transcription kit from Promega. The dsRNA was precipitated with ethanol, dissolved in 300ul of pure water, denatured by heating to 65C for 30min and re-annealed by slow cooling to room temperature.2x10^6cells were treated with 50ug of dsRNA in 25cm2 T-flask for 1 hour in serum-free Schneider?s media. They were further brought up 6ml of Schneider?s media supplemented with 10% FBS and grown to confluence (~4 days). The cells were passaged to a 75cm2 T-flask and treated the second time with 200ug of dsRNA as described above. Following the treatment the cells were grown to confluence in 15ml Schneider?s media. 1ml of resulted culture was taken for preparation of nuclear and cytoplasmic protein extracts and the rest was in vivo crosslinked with formaldehyde. The protocol typically yields about 7x10^7 crosslinked cells, which is enough for about 7 ChIPs. And the RNAi treated cells were treated with formaldehyde as in normal cultured cell.
After treatment with formaldehyde, Drosophila tissue culture cells were lysed with SDS and sonication (on ice) in the presence of protease inhibitors. The resulting chromatin was treated with non-ionic detergents at physiological concentrations of monovalent cations. Ribonucleic acid was removed by RNAse A, and the DNA was purified using SDS, proteinase K, and organic extraction, followed by ethanol precipitation.
1. Add 30ml of PAS (50% suspension in RIPA (-PMSF)) to 500ml of crosslinked chromatin. Incubate 1h at +40C. 2. Spin suspensions for 2min at top speed +40C. Transfer supernatants to new tubes. Add 5ml of 100mM PMSF solution in isopropanol to each 500ml aliquot of precleared chromatin. 3. Add appropriate amount of antibody to each reaction. Do not forget to set up no Ab control. Incubate for 15 hours at +40C on rotating weel. 4. Add 40ml of PAS (50% suspension in RIPA (-PMSF)), incubate 3h at +40C on rotating weel. 5. Wash the beads 5 times 10min each with 1ml of RIPA, then one time with 1ml of LiCl ChIP buffer and finally twice with 1ml of TE. To pellet the beads between washes spin samples for 20sec +40C at top speed. Do all the washes at +40C. 6. Resuspend the beads in 100ml TE add 1ml (final 50mg/ml) of RNAse A (10mg/ml) incubate 30min at +370C. 7. Add 7.5ml (final 0.5%) of 10% SDS and 3.8ml (final 0.5mg/ml) of Proteinase K (20mg/ml). Incubate overnight at +370C. 8. Transfer samples at +650C, incubate 6h. 9. Add 4.5ml of 5M NaCl (140mM final). Extract samples with 150ml of phenol-chloroform by vortexing for 30 sec, centrifuge for 10 min at RT, take 120ml of aqueous phase, back-extract organic phase with 150ml of TEN 140 (10mM Tris-HCl pH8.0; 1mM EDTA; 140mM NaCl). Take 150ml of aqueous phase. Combine aqueous phases (you will get 120ml + 150ml=270ml of solution). 10. Extract samples with 300ml of chloroform by vortexing for 30 sec, centrifuge for 5 min at RT. Transfer the upper aqueous phase into the new tube. Add 30ml of 3M NaAc pH 5.0 and 2ml of glycogen (5mg/ml) to aqueous phase. Precipitate DNA with 900ml of EtOH at -700C for 1h. 11. Spin for 10min, top speed at +40C. Wash the pellet in 300ml of 70%EtOH 12. Spin for 10min, top speed at +40C. If you plan to do qPCR analysis only dissolve the pellet in 150ml of pure H2O. If you plan to do both qPCR and microarray hybridization then first dilute DNA pellets in 12ml of pure H2O transfer 4ml of DNA solution to a new eppendorf tube and add 46ml of pure H2O. Use the latter for qPCR and the former for subsequent amplification and labeling. Store DNA solutions at -200C. 13. Prepare dilutions of DNA isolated from the original crosslinked chromatin (also called ?Input DNA? or simply ?Input?) following the chart below. Use the stock with concentration of 0.5% of input DNA per ml of solution (see: ?Isolation of ChIP Input DNA? protocol).
|
| Label |
biotin
|
| Label protocol |
1. Prehybridize array for 1 hr in 200ml of 1xMES-Triton at 450C with 45 rpm rotation. 2. Incubate hybridization cocktail for 10min at 1000C then 10min at 450C. Spin at max speed for 3 min at RT. Transfer the supernatant to new tube. Spin for 3 more minutes use 200ml of resulted supernatant for hybridization. 3. Hybridize 18hr at 450C with 45 rpm rotation. 4. Use fluidics station EukGE-WS2v4 protocol (Affymetrix) for washing and staining.
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| |
|
| Hybridization protocol |
1. Prehybridize array for 1 hr in 200ml of 1xMES-Triton at 450C with 45 rpm rotation. 2. Incubate hybridization cocktail for 10min at 1000C then 10min at 450C. Spin at max speed for 3 min at RT. Transfer the supernatant to new tube. Spin for 3 more minutes use 200ml of resulted supernatant for hybridization. 3. Hybridize 18hr at 450C with 45 rpm rotation. 4. Use fluidics station EukGE-WS2v4 protocol (Affymetrix) for washing and staining.
|
| Scan protocol |
Standard Affymetrix Array Scanning Protocol was used
|
| Description |
channel ch1 is input DNA;
|
| Data processing |
M-value normalization protocol. The log-intensity ratio values (M-values) are calculated for all perfect match (PM) probes as log2(ChIP intensity) - log2(input intensity). The M values are then shifted so that the mean is equal to 0.
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| |
|
| Submission date |
Oct 07, 2011 |
| Last update date |
Oct 13, 2011 |
| Contact name |
DCC modENCODE |
| E-mail(s) |
[email protected]
|
| Phone |
416-673-8579
|
| Organization name |
Ontario Institute for Cancer Research
|
| Lab |
modENCODE DCC
|
| Street address |
MaRS Centre, South Tower, 101 College Street, Suite 800
|
| City |
Toronto |
| State/province |
Ontario |
| ZIP/Postal code |
M5G 0A3 |
| Country |
Canada |
| |
|
| Platform ID |
GPL6629 |
| Series (1) |
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