 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jul 29, 2009 |
| Title |
S2-R1 extraction1_array1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
S2-R1 extraction1_array1 channel_1
|
| Organism |
Drosophila melanogaster |
| Characteristics |
cell line: S2-DRSC
tissue: embryo-derived cell-line
sex: Male
|
| Growth protocol |
Cell growth protocol; Grow 10ml of cells in 100mm dishes For Kc and S2 cells use : Schneider's medium (invitrogen 11720-067) 10% ModEncode serum 1% Penicillin/Streptomycin/Glutamine (PSG) (Invitrogen 10378-016) small edit For ML-DmBG3-c2 cells use: Schneider's medium 10% ModEncode serum 1% PSG 10ug/ml human insulin (sigma I9278)) Cells are grown at 25C. Cells are happiest between 1.5X106 and 2.0X106
Brdu labeling timing protocol; For Replication Timing Experiments: 1.Cells are counted and split to 1.5X10^6/ml (approximately 10 ml) 2.Set up two populations of cells- early timing and late timing populations 3.Early population is treated with 1mM Hydroxyurea (HU; Sigma H8627) for 3h, then 5-Bromo-2-deoxyuridine (BrdU; Roche 280-879) is added to 50ug/ml final concentration and incubated for 21hours 4.Late population is treated with 1mM HU for 24 hours. 5.The next day, both populations are washed with warm serum free medium and resuspended in medium with serum 6.The early population receives another 50ug/ml BrdU for 1hour. Cells are harvested. 7.The late population incubates for 3-4 hours without drug. At this time, 50ug/ml BrdU is added and allowed to incubate for 2 hours. Harvest cells. 8.Wash cells with 1X PBS and prep DNA, or freeze pellet at -20C
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA isolation protocol; 1.Grow 10ml of cells to 1.5 x 106 in 100mm dishes 2.Harvest cells into 15ml conical tubes. 3.Pellet cells at 1000g for 5 minutes 4.Wash cells once in cold 1X PBS 5.Resuspend pellet in 0.7ml 10mM Tris pH 9.5 6.Add 0.7ml NDS. Invert to mix 7.Add 0.1ml 20mg/ml Proteinase K (Amresco 0706-1G). Invert to mix 8.Inc. 37C, 2 hours. 9.Add 1.5ml 1X TE. Invert to mix 10.Add 3.0ml phenol/chloroform. Invert tubes over 5 minutes 11.Centrifuge 3500 rpm for 10-12 minutes 12.Remove top layer with cut p1000 tips to a new 15ml tube 13.Repeat steps 10-12. 14.Add 2.5 volumes of room temp. 100% Ethanol. Invert 15.Inc. at room temp overnight 16.Centrifuge tubes 3500-3700rpm for 10-12 minutes 17.Wash pellet with 70% Ethanol and transfer pellet to a 1.5ml tube 18.Centrifuge full speed for 5 minutes 19.Air dry pellet 20.Resusp. pellet in 250ul 1X TE 21.Add 5ul 10mg/ml RNaseA (Amresco 0675-250mg). Inc. 37C for 1h 22.Add 1/10 volume 3M NaOAc. Vortex 23.Add 2X volume cold 100% Ethanol. Vortex 24.Inc. -80C 20-30 minutes 25.Centrifuge at 4C for 15 minutes 26.Wash with 70% Ethanol 27.Air dry pellet. Resusp. in 250ul 1X TE 28.Spec DNA Shearing DNA 29.Shear DNA 3X 10 seconds with 1minute on ice in between 30.Run 5ug on 1% agarose gel 31.Set up Southern overnight 32.Check for BrdU incorporation by western. anti-BrdU antibody (BD BioSciences 555627 ) is 1:1000 dilution in milk/TBS-T
Brdu IP protocol; Couple Antibody to beads 1.Aliquot 50ul of IgG sheep anti-mouse magnetic beads (Invitrogen) per experiment to a 1.5ml tube 2. Wash 3X with 1ml of 1XPBS+5mg/ml BSA . PBS/BSA is filter sterilized and at 4C Washes are done using a magnetic concentrator MPC-S (invitrogen) and supernatant is removed using vaccuum fitted with 26g needle. 3.Resusp beads in 1ml PBS/BSA 4.Add 5ul anti-BrdU antibody (BD BioSciences) 5.Incubate on labquake rotator, overnight, 4C Couple DNA to beads 6.Wash beads 3X with 1ml PBS/BSA as above. 7.Resusp in approx. 55ul of PBS/BSA. Keep beads on ice 8.Prepare DNA. Need 15-25ug DNA/experiment 9.Aliquot DNA into 1.5ml tubes 10.Inc. 100C for 5 minutes 11.Place on ice 12.Add 450ul RIPA buffer to DNA 13.Add 50ul of beads. Invert to mix 14.Incubate on labquake rotator, overnight, 4C Recover bound DNA 15.Place tubes in magnetic concentrator 16.Save 100ul as INPUT. Freeze at -20C 17.Remove sup with 26g needle 18.Wash 4X with cold RIPA buffer. Resusp for 5 minutes on room temp labquake after each wash 19.Wash 1X with room temp 1X TE 20.Resusp in 150ul 1XTE+1%SDS 21.Inc in 65C waterbath for 10 minutes. Vortex samples 2-3X during incubation. 22.Add 150ul TE/glycogen/PK. Inc 37C 1hour 23.Centrifuge briefly and aliquot supernatant to a new 1.5ml tube 24.Add 12ul 5M NaCl. invert to mix 25.Add 300ul phenol/chloroform. Invert over 5 minutes 26.Centrifuge 5-10 minutes 27.Remove top layer to a new tube 28.Add 700ul cold 100% ethanol. Invert 29.Inc -80 for 1hour 30.Centrifuge for 15 minutes at 4C 31.Wash with 70% ethanol and spin for 5 minutes 32.Air dry pellet and resusp in 15ul of DNase/Rnase free water (Ambion) 33.Spec DNA. Can store at -20C
|
| Label |
Cy5
|
| Label protocol |
Sequenase labeling protocol; 1.Thaw DNA samples on ice 2.Transfer to 0.2ml pcr tubes. Open tubes and place inside a 1.5ml tube 3.Speed vac samples on medium heat for approx. 15-18 minutes 4.Resusp DNA in sequenase primer mix: 5X sequenase buffer 2ul (US Biochemicals) random nonamer 5ul (NNNNNNNNN; hand mixed 25,25,25,25) Ambion dH2O 1ul 5.Thaw Cy dyes (Cy 3 dUTP and Cy 5 dUTP; Perkin Elmer) on ice and in the dark 6.Add 2ul of cy dye to DNA sample 7.Start pcr program: 95C 3' 4C 2' 37C 30' ramp 95C 2' 4C 2' 37C 30' ramp 4C hold 8.At the first 4C step in program, add the following: 5X sequenase buffer 1ul Low T 1.5ul (5ul dATP, 5ul dCTP, 5ul dGTP, 2ul dTTP, 83ul ambion dH2O) 100 mM BSA 1ul 100mM DTT 0.75ul sequenase 1ul 9.At the second 4C step, add additional sequenase and finish program sequenase buffer DLT 0.7ul sequenase 0.3ul 10.Clean samples on YM-30 microcon column (Millipore) according to directions 11.Spec samples
|
| |
|
| Channel 2 |
| Source name |
S2-R1 extraction1_array1 channel_2
|
| Organism |
Drosophila melanogaster |
| Characteristics |
cell line: S2-DRSC
tissue: embryo-derived cell-line
sex: Male
|
| Growth protocol |
Cell growth protocol; Grow 10ml of cells in 100mm dishes For Kc and S2 cells use : Schneider's medium (invitrogen 11720-067) 10% ModEncode serum 1% Penicillin/Streptomycin/Glutamine (PSG) (Invitrogen 10378-016) small edit For ML-DmBG3-c2 cells use: Schneider's medium 10% ModEncode serum 1% PSG 10ug/ml human insulin (sigma I9278)) Cells are grown at 25C. Cells are happiest between 1.5X106 and 2.0X106
Brdu labeling timing protocol; For Replication Timing Experiments: 1.Cells are counted and split to 1.5X10^6/ml (approximately 10 ml) 2.Set up two populations of cells- early timing and late timing populations 3.Early population is treated with 1mM Hydroxyurea (HU; Sigma H8627) for 3h, then 5-Bromo-2-deoxyuridine (BrdU; Roche 280-879) is added to 50ug/ml final concentration and incubated for 21hours 4.Late population is treated with 1mM HU for 24 hours. 5.The next day, both populations are washed with warm serum free medium and resuspended in medium with serum 6.The early population receives another 50ug/ml BrdU for 1hour. Cells are harvested. 7.The late population incubates for 3-4 hours without drug. At this time, 50ug/ml BrdU is added and allowed to incubate for 2 hours. Harvest cells. 8.Wash cells with 1X PBS and prep DNA, or freeze pellet at -20C
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA isolation protocol; 1.Grow 10ml of cells to 1.5 x 106 in 100mm dishes 2.Harvest cells into 15ml conical tubes. 3.Pellet cells at 1000g for 5 minutes 4.Wash cells once in cold 1X PBS 5.Resuspend pellet in 0.7ml 10mM Tris pH 9.5 6.Add 0.7ml NDS. Invert to mix 7.Add 0.1ml 20mg/ml Proteinase K (Amresco 0706-1G). Invert to mix 8.Inc. 37C, 2 hours. 9.Add 1.5ml 1X TE. Invert to mix 10.Add 3.0ml phenol/chloroform. Invert tubes over 5 minutes 11.Centrifuge 3500 rpm for 10-12 minutes 12.Remove top layer with cut p1000 tips to a new 15ml tube 13.Repeat steps 10-12. 14.Add 2.5 volumes of room temp. 100% Ethanol. Invert 15.Inc. at room temp overnight 16.Centrifuge tubes 3500-3700rpm for 10-12 minutes 17.Wash pellet with 70% Ethanol and transfer pellet to a 1.5ml tube 18.Centrifuge full speed for 5 minutes 19.Air dry pellet 20.Resusp. pellet in 250ul 1X TE 21.Add 5ul 10mg/ml RNaseA (Amresco 0675-250mg). Inc. 37C for 1h 22.Add 1/10 volume 3M NaOAc. Vortex 23.Add 2X volume cold 100% Ethanol. Vortex 24.Inc. -80C 20-30 minutes 25.Centrifuge at 4C for 15 minutes 26.Wash with 70% Ethanol 27.Air dry pellet. Resusp. in 250ul 1X TE 28.Spec DNA Shearing DNA 29.Shear DNA 3X 10 seconds with 1minute on ice in between 30.Run 5ug on 1% agarose gel 31.Set up Southern overnight 32.Check for BrdU incorporation by western. anti-BrdU antibody (BD BioSciences 555627 ) is 1:1000 dilution in milk/TBS-T
Brdu IP protocol; Couple Antibody to beads 1.Aliquot 50ul of IgG sheep anti-mouse magnetic beads (Invitrogen) per experiment to a 1.5ml tube 2. Wash 3X with 1ml of 1XPBS+5mg/ml BSA . PBS/BSA is filter sterilized and at 4C Washes are done using a magnetic concentrator MPC-S (invitrogen) and supernatant is removed using vaccuum fitted with 26g needle. 3.Resusp beads in 1ml PBS/BSA 4.Add 5ul anti-BrdU antibody (BD BioSciences) 5.Incubate on labquake rotator, overnight, 4C Couple DNA to beads 6.Wash beads 3X with 1ml PBS/BSA as above. 7.Resusp in approx. 55ul of PBS/BSA. Keep beads on ice 8.Prepare DNA. Need 15-25ug DNA/experiment 9.Aliquot DNA into 1.5ml tubes 10.Inc. 100C for 5 minutes 11.Place on ice 12.Add 450ul RIPA buffer to DNA 13.Add 50ul of beads. Invert to mix 14.Incubate on labquake rotator, overnight, 4C Recover bound DNA 15.Place tubes in magnetic concentrator 16.Save 100ul as INPUT. Freeze at -20C 17.Remove sup with 26g needle 18.Wash 4X with cold RIPA buffer. Resusp for 5 minutes on room temp labquake after each wash 19.Wash 1X with room temp 1X TE 20.Resusp in 150ul 1XTE+1%SDS 21.Inc in 65C waterbath for 10 minutes. Vortex samples 2-3X during incubation. 22.Add 150ul TE/glycogen/PK. Inc 37C 1hour 23.Centrifuge briefly and aliquot supernatant to a new 1.5ml tube 24.Add 12ul 5M NaCl. invert to mix 25.Add 300ul phenol/chloroform. Invert over 5 minutes 26.Centrifuge 5-10 minutes 27.Remove top layer to a new tube 28.Add 700ul cold 100% ethanol. Invert 29.Inc -80 for 1hour 30.Centrifuge for 15 minutes at 4C 31.Wash with 70% ethanol and spin for 5 minutes 32.Air dry pellet and resusp in 15ul of DNase/Rnase free water (Ambion) 33.Spec DNA. Can store at -20C
|
| Label |
Cy3
|
| Label protocol |
Sequenase labeling protocol; 1.Thaw DNA samples on ice 2.Transfer to 0.2ml pcr tubes. Open tubes and place inside a 1.5ml tube 3.Speed vac samples on medium heat for approx. 15-18 minutes 4.Resusp DNA in sequenase primer mix: 5X sequenase buffer 2ul (US Biochemicals) random nonamer 5ul (NNNNNNNNN; hand mixed 25,25,25,25) Ambion dH2O 1ul 5.Thaw Cy dyes (Cy 3 dUTP and Cy 5 dUTP; Perkin Elmer) on ice and in the dark 6.Add 2ul of cy dye to DNA sample 7.Start pcr program: 95C 3' 4C 2' 37C 30' ramp 95C 2' 4C 2' 37C 30' ramp 4C hold 8.At the first 4C step in program, add the following: 5X sequenase buffer 1ul Low T 1.5ul (5ul dATP, 5ul dCTP, 5ul dGTP, 2ul dTTP, 83ul ambion dH2O) 100 mM BSA 1ul 100mM DTT 0.75ul sequenase 1ul 9.At the second 4C step, add additional sequenase and finish program sequenase buffer DLT 0.7ul sequenase 0.3ul 10.Clean samples on YM-30 microcon column (Millipore) according to directions 11.Spec samples
|
| |
|
| |
| Hybridization protocol |
Agilent hybridization protocol; Setting up hybridization 1.Thaw samples on ice, in the dark 2.Set up hyb reaction: (Oligo aCGH/ChOP-on-Chip Hyb kit; Agilent) sample 25ul Ambion dH2O 175ul 10X Blocking Solution 50ul 2X Hybridization buffer 250ul 3.Inc 95C, 5 minutes 4.Spin down briefly and keep at 37C while getting slide ready 5.Place gasket with agilent side facing up on metal chamber 6.Pipet 490ul of hyb reaction on gasket 7.Lay down microarray on hyb reaction with Agilent side down. 8.Assemble metal chamber 9.inc. 16-20 hours in rotating hyb oven at 65C Washing microarrays 10.Dissamble arrays in tip box lid containing Buffer 1 (Agilent) 11.Wash slide in buffer 1 with stirring, 5 minutes 12.Wash slide in buffer 2 with stirring, 5 minutes 13.Wash slide in acetonitrile, 30 seconds 14.Wash slide in buffer 3, 30 seconds 15.Store in light proof holder and scan
|
| Scan protocol |
Array scanning protocol; Scanning per Agilent recommendations.
|
| Description |
N/A
|
| Data processing |
Array Data normalization protocol; Arrays are normalized using the Limma package for R. RG<-read.maimages(target,source='agilent') RGb<-backgroundCorrect (RG, method='none') MA<-normalizeWithinArrays(RGb, method='loess') Normalized data is output in with the following columns Row: y pos on array Col: x pos on array Start: ? Sequence: probe sequence ProbeUID: probe number on array ControlType: type of control. experimental values are 0 ProbeName: internal database id GeneName: internal database id SystematicName: internal database id Description: Chr pos and location M..X1: M value for experiment 1 A..X1: A value for experiment 1 Processed data are obtained using following parameters: genome version is r5
|
| |
|
| Submission date |
Jul 23, 2009 |
| Last update date |
Jul 29, 2009 |
| 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 |
GPL7787 |
| Series (1) |
|
| Supplementary file |
Size |
Download |
File type/resource |
| GSM432697_MacAlpine_251663510021_S01_CGH-v4_95_Feb07.txt.gz |
68.6 Mb |
(ftp)(http) |
TXT |
| Processed data are available on Series record |
|
|
|
|
 |
