NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM1085935 Query DataSets for GSM1085935
Status Public on Feb 22, 2013
Title HP1 wa191 Nuclear L3 Su(var) 3-9 06_17 D.melanogaster Input Rep1
Sample type genomic
 
Source name HP1 wa191 Nuc. L3 Su(var) Input expt.1388 channel_1
Organism Drosophila melanogaster
Characteristics strain: Su(var) 3-9 06 17 mutant
developmental stage: 3rd Instar Larvae
genotype: Su(var)3-9^06/Su(var)3-9^17
transgene: n/a
Extracted molecule genomic DNA
Extraction protocol Flies are grown in population cages. After the larvae mature into flies in the inoculated tubs, the flies are transferred to the population cages. The adult flies are raised over a 22-day cycle, with cycles overlapping so that when one adult population has become less productive, another is ready to take its place.
Growth conditions and collection protocol to harvest third instar larvae from bottles.
Drosophila tissues were homogenized, and treated with formaldehyde. Nuclei were lysed with SDS and sonication in the presence of protease inhibitors. The resulting chromatin was treated with non-ionic detergents at physiological concentrations of monovalent cations.
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.
 
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 Served as Input for: 1412.Mvalues.bedgraph
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.
 
Submission date Feb 21, 2013
Last update date Mar 12, 2013
Contact name DCC modENCODE
E-mail(s) help@modencode.org
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)
GSE44504 HP1 wa191.D.mel 3rd Instar Larvae Nuclei Su(var) 3-9 06_17 Mutant

Supplementary file Size Download File type/resource
GSM1085935_pirrotta_1683_L3_Su_var_3-9_06_17_1_Input_20cyc.1388.CEL.gz 26.6 Mb (ftp)(http) CEL
Processed data are available on Series record

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap