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Sample GSM551131 Query DataSets for GSM551131
Status Public on Nov 02, 2010
Title ATRX_29-1-7_rep1
Sample type genomic
 
Channel 1
Source name Cultured Primary Human Erythroblasts
Organism Homo sapiens
Characteristics cell type: Cultured Primary Human Erythroblasts
antibody: ATRX H300 (Santa Cruz Biotechnology, batch G0105)
Growth protocol Basic Protocol 1: Preparation of Peripheral Blood Mononuclear Cells for Erythroid Cultures Cultures can be initiated with cells derived from a whole unit of blood obtained from normal donors (or polycythemic patients undergoing phlebotomy). The starting material, i.e., the “buffy-coat” fraction, is readily available, since it is routinely separated and discarded by the blood bank during preparation of packed red blood cells (RBC) and platelet-rich plasma. Anemic patients commonly show high frequencies of erythroid progenitors; cultures from anemic patients (e.g., thalassemia, sickle cell anemia) are usually initiated with cells derived from ~20 ml peripheral blood. Materials * Peripheral blood or buffy coat fraction * Phosphate-buffered saline (PBS), pH 7.4 (appendix 2A) * Ficoll-Hypaque (density = 1.077 g/ml) * 15- or 50-ml centrifuge tubes * Centrifuge 1. Dilute peripheral blood or the buffy coat fraction 1:1 (v/v) with PBS. Gently layer on top of a layer of Ficoll-Hypaque such that the Ficoll-Hypaque layer constitutes one-third of the total volume. Depending on the blood volume, use either 15- or 50-ml tubes. For convenience, tubes (available from Sigma) containing a special grid that separates the blood from the Ficoll can be used. 2. Centrifuge 20 min at 400 × g, at room temperature. Do not apply the brake. 3. Aspirate off the top layer (consisting of plasma and platelets), collect the interphase layer of mononuclear cells with a pipet, and transfer to a 50-ml tube. 4. Add 40 ml PBS, mix, then centrifuge 5 min at 160 × g, room temperature. Aspirate the supernatant, then resuspend the pellet in the residual buffer by tapping. 5. Add 40 ml PBS, mix, then centrifuge 5 min at 100 × g, room temperature, and aspirate the supernatant. Repeat this procedure for an additional wash. Use low-speed centrifugation in order to remove residual platelets. All procedures should be performed at room temperature. If the blood cannot be processed on the same day, leave overnight at room temperature. In experiments where Hb content is measured, special care has to be taken to avoid contamination by mature RBC that originated from the processed blood. This does not usually present a problem when normal blood is processed, but may occur with blood derived from patients, e.g., with thalassemia. The contaminating RBCs can be of patient origin and/or derived from transfused blood. In both cases, they may interfere with measurement of the Hb produced in vitro. When the interphase layer is heavily contaminated with RBC, the latter cells should be removed by osmotic hemolysis (see Support Protocol 6) prior to culturing. When only slightly contaminated, the mononuclear cells may be cultured without hemolysis; RBC tend to lyse and disappear during culture. Alternatively, they may be removed by Percoll separation at the second phase of the culture (see Support Protocol 7). [Top of Page] Basic Protocol 2: Erythroid Cell Culture Procedure The culture procedure is divided into two phases. The first is an EPO-independent phase in which peripheral blood mononuclear cells are isolated (described in Basic Protocol 1) and cultured in the presence of a combination of growth factors, but in the absence of EPO. During this phase, early erythroid-committed progenitors, called burst-forming units erythroid (BFUe), proliferate and differentiate into colony-forming unit erythroid (CFUe)–like progenitors. In the second phase, these cells, cultured in an EPO-supplemented medium, continue to proliferate and mature into orthochromatic normoblasts and enucleated erythrocytes. Materials * Complete MEM medium (see recipe) * Preselected FBS (see recipe); do not heat-inactivate * 50 mg/ml cyclosporin A (Sandoz) * Conditioned medium from 5637 human bladder carcinoma cell line (see recipe) * Mononuclear cells, washed (see Basic Protocol 1) * 10% (w/v) deionized BSA (see recipe) * 2-mercaptoethanol (Sigma) * 4 mg/ml dexamethasone sodium phosphate (Elkin-Sinn Pharmaceuticals, Cherry Hill N.J.) * Human holo-transferrin (Sigma) * Human recombinant stem cell factor (CytoLab Ltd.; http://www.cytolab.com/) * Human recombinant erythropoietin (EPO; Ortho Pharmaceutical) * 25- or 75-cm2 tissue culture flasks 1. Prepare Phase I medium by adding the following ingredients to complete MEM medium: * 10% (v/v) preselected FBS (not heat-inactivated) * 1 µg/ml cyclosporin A (add from 50 mg/ml stock) * 10% (v/v) conditioned medium from 5637 human bladder carcinoma cell line. 2a. For cells obtained from peripheral blood (10 to 20 ml): Plate the washed mononuclear cells in Phase I medium in 25-cm2 flasks at a culture volume equivalent to the original volume of blood. 2b. In the case of cells obtained from a whole blood unit (400 to 500 ml): Plate the washed mononuclear cells in Phase I medium in two 75-cm2 flasks at a culture volume of 80 to 100 ml. 3. Incubate the cultures at 37°C in a humidified, 5% CO2 incubator for 5 to 7 days. Full humidification is achieved by leaving a tray with water standing at the bottom of the incubator. 4. After the 5- to 7-day incubation in phase I, shake the cultures and harvest the nonadherent cells (do not trypsinize). 5. Centrifuge the cells 5 min at 160 × g, room temperature, and aspirate the supernatant. Fill the tube with complete MEM, centrifuge 5 min at 160 × g, room temperature, and aspirate the supernatant. Repeat for a second wash. 6. Prepare Phase II medium by adding the following ingredients to complete MEM medium: * 30% (v/v) preselected FBS (not heat-inactivated) * 1% (w/v) deionized BSA (add from 10% w/v stock) * 10–5 M 2-mercaptoethanol * 10–6 dexamethasone (add from 4 mg/ml stock) * 0.3 mg/ml human holo-transferrin * 10 ng/ml human recombinant stem cell factor * 1 U/ml human erythropoietin (EPO). 7. Plate cells in the original volume of phase II medium (as in step 2a or 2b according to the original blood volume) and incubate up to 14 days at 37°C in a humidified, 5% CO2, incubator.
Extracted molecule genomic DNA
Extraction protocol Protocol: Chromatin Immunoprecipitation coupled with Microarray Analysis (ChIP-chIP): I. Dynabead Preparation -Make PBS/BSA -For each ChIP reaction. Wash 100ul of beads 3-4 times with PBS BSA (5mg/ml) -Transfer beads to a 500ul PCR tube. -For ATRX antibody binding, Add 200ul ATRX H300, 22ul 50mg/ml BSA, 4ul ssDNA (Invitrogen 10mg/ml) -For NoAb Rxn. Add 222ul PBS BSA 4ul ssDNA -Incubate 3-4 hrs on roller at 4 oC -Wash 4 times in PBS BSA -Resuspend in 100ul PBSBSA and add 4ul (40ug) ssDNA For each preclear reaction -Take 100ul of Dynabeads -Wash 3-4 times with 1ml PBS BSA (5mg/ml freshly prepared) -Add 100ul of PBS BSA(5ug/ul) -Add 4ul ssDNA (10ug/ul) -Incubate at 4oC on roller with antibody binding reactions -Use 100ul for each preclearing reaction II. Cell cross-linking: Need 4oC PBS, Formaldehyde, EGS 1M glycine, Trypan blue, gilsons, timer 1.5ml tubes, calculator, A. Suspension Cells: 1. Make up EGS (Pierce 21565) stock fresh: 0.046g EGS in 200ul DMSO for 500mM stock 2. Chill PBS 3. Wash twice in PBS and resuspend in 20mls PBS (Important as FCS inhibits EGS) 4. Count cells 5. Use 3x107 – 5x107 cells for each ChIP reaction 6. Add EGS (it will form a white PPT which disperses afer a second) to 2mM and incubate at room temperature of 45 mins with occasional mixing. (80ul per 20mls of PBS). 7. Add formaldehyde solution to 1% for 20mins (526ul /20mls. 4 x 131.5ul) 8. Add 1/8 volume of 1M glycine to tubes to quench formaldehyde.(2.5ml glycine / 20ml PBS). 9. Spin cells at 1k for 5 min at 4oC. Make up protease inhibitors and add to lysis buffers. 10.Wash twice with 20ml 4oC PBS III. Cell sonication (keep all steps on ice): Note: Add protease inhibitors to all lysis buffers before use. If using a protease inhibitor cocktail tablet from Complete, dissolve one tablet in 2 ml water for 25X solution. Store aliquots at -20oC. 1. Resuspend each tube of cells in 10 ml of lysis buffer I. Rock at 4oC for 10 min, and then spin at 2k in a table-top centrifuge for 2 min at 4oC. 2. Resuspend each tube of cells in 5-10 ml of lysis buffer 2. Pellet nuclei in table-top centrifuge by spinning at 2.5k for 2 min at 4oC. 3. Resuspend the pellet in each tube in 3 ml lysis buffer 3. (Retain the buffer for step 6) 4. Sonicate the suspension in 15ml corning blue top tubes. 2mls per tube. Use the Bioruptor at high power for 20mins (4x5mins). After each 5 minute pulse, replenish the ice. 5. Add 1/10 volume of 10% Triton X-100. Transfer to 1.5 ml centrifuge tubes. Spin out debris at 14k for 10 min at 4oC. The lysate contains the fragmented DNA. 6. Save pellet for western analysis and take 50ul lysate for western analysis 7. Transfer lysate to 15 ml conical tube, adjusting the volume to 3 ml using lysis buffer 3. 8. Save 50µl of cell lysate from each sample as input. Save 50ul to assess sonication efficiency. IV. Chromatin immunoprecipitation: -Add 100ul of the preclearing beads from step I to the 3mls of chromatin lysate -Preclear on roller at 4 oC for 1 hour -Precipitate beads from chromatin using magnet and remove chromatin to new 3ml tubes -Add the washed antibody bound beads from step 1 -Incubate overnight on roller at 4 oC V. Washing, eluting, and reverse cross-linking: Note: Carry steps 1-3 in the cold room and all wash solution must be ice cold. 1. Transfer lysates from the 3 ml tubes to a 1.5ml tube. 2. Wash the IPs 5 times with 1 ml wash buffer (be sure to add protease inhibitors to the wash buffer). Use the magnetic stand to precipitate the beads. 3. Wash once with 1 ml cold PBS. Aspirate residual PBS. 4. Add 250µl of elution buffer. Elute on a roller at room temperature for 15 minutes. 5. Add 250ul more of elution buffer to beads, vortex, precipitate and combine with previous round of elution. 6. Take 50ul of eluate for western blot analysis 7. PPT beads and remove eluate to a new tube. Add 20ul 5M NaCl and incubate at 65oC for 4 hours. Thaw input, make up to 500ul with elution buffer, add 20ul 5M NaCl and incubate for 4hrs at 65oC. VI. RNase, Proteinase K, Phenol/Chloroform, EtOH PPT: Remove beads. Then add 20ul of 1M Tris pH6.5, 10ul 0.5M EDTA, 2ul 10mg/mg Proteinase K. Incubate for 1hrs at 45°C. Phenol chloroform and EtOH PPT with 20ug of carrier glycogen. Resuspend in 21ul sigma water. Use low volume to give the option of labelling for ChIP on Chip. For real time PCR, use 1/12 ul per reaction Solutions PBS BSA 5mg/ml For 50mls: 5mls 10XPBS, 250mg BSA Fraction V, Filter sterilise. Make up fresh every time. Lysis Buffer 1 (100 ml): 100mM HEPES 140mM NaCl 1mM EDTA 10% Glycerol 0.5% NP40 0.25% Triton X Lysis Buffer 2 (100 ml): 200mM NaCl 1mM EDTA 0.5mM EGTA 10mM Tris pH 8 Lysis Buffer 3 (100 ml): 1mM EDTA 0.5mM EGTA 1mMTris-HCl pH8 100mM NaCl 0.1% Na DOC 0.5% N-lauroyl Sarcosine Wash buffer (RIPA Buffer, 100 ml): 50mM Hepes, pH 7.6 1mM EDTA 0.7% Na DOC 1% NP40 500mM LiCl
Label Cy3
Label protocol Random Labelling DNA for array CGH Equipment and reagents • BioPrime Labelling Kit (Invitrogen 18094-011) • 10X dNTP mix (Amersham 27-2035-01) • 1mM Cy3-dCTP (Amersham PA53021) • 1mM Cy5-dCTP (Amersham PA55021) • HPLC water BDH 152736D • Micro-spin G50 columns (Amersham, 275330-01) Method 1. Random label 450 ng of DNA in a final reaction volume of 150 μl by mixing the DNA with 60 μl 2.5X Random Primers Solution (from BioPrime Labelling Kit) and make up to 130 μl with HPLC water. Make 1 reaction for ChIP DNA and 1 reaction for input DNA. 2. Denature DNA in a heat block for 10 min at 100°C, and immediately cool on ice. 3. The following reagents are added on ice: 15 μl 10X dNTP mix (so prepared: dNTPs Amersham 27-2035-01 4x25umol C 1μl , A 2 μl, G 2 μl , T 2 μl , HPLC water 93 μl) 1.5 μl Cy3 (for ChIP DNA) or Cy5 (for input DNA) labelled dCTP (1 mM) from Amersham PA53021 and PA55021 3 μl Klenow Fragment from Biopreime Labelling Kit Mix gently but thoroughly. 4. Incubate the reaction @ 37°C overnight in darkness and then stop it by adding 15 μl of stop buffer (from BioPrime Labelling kit). Removal of Labelled Nucleotides from DNA Labelling Reactions (The recommended volume for application to a single column is 50 μl, you therefore have to use 3 columns per 150 µl labelling reaction) Column Preparation 1. Resuspend the resin in the columns by gentle vortexing. 2. Loosen the cap one-fourth turn and snap off the bottom closure. 3. Place the columns in a 1.5 ml screw-cap microcentrifuge tube for support. Alternatively, cut the cap from a flip-top tube and use this tube for support. 4. Pre-spin the columns for 1 minute at 2700 rpm. DO NOT pulse as this will override the variable speed setting. Wash columns applying 50 mcl of HPLC water to the resin and spin 1 min at 2700 rpm. Use columns immediately after preparation to avoid the resin drying out. Sample Application 1. Place the columns in a new 1.5 ml tube and slowly apply 50 μl to the centre of the angled surface of the compacted resin bed of each of the columns, being careful not to disturb the resin bed. Careful application of the sample to the centre of the bed is essential for good separation. Do not allow any of the samples to flow around the sides of the bed. 2. Spin the columns for 2 minutes at 2700 rpm. The purified samples are collected in the bottom of the support tube. 3. Discard the columns and retain the flow-through samples. 4. Combine the three samples and run 5 μl on a 1% agarose gel
 
Channel 2
Source name Cultured Primary Human Erythroblasts
Organism Homo sapiens
Characteristics cell type: Cultured Primary Human Erythroblasts
antibody: none
Growth protocol Basic Protocol 1: Preparation of Peripheral Blood Mononuclear Cells for Erythroid Cultures Cultures can be initiated with cells derived from a whole unit of blood obtained from normal donors (or polycythemic patients undergoing phlebotomy). The starting material, i.e., the “buffy-coat” fraction, is readily available, since it is routinely separated and discarded by the blood bank during preparation of packed red blood cells (RBC) and platelet-rich plasma. Anemic patients commonly show high frequencies of erythroid progenitors; cultures from anemic patients (e.g., thalassemia, sickle cell anemia) are usually initiated with cells derived from ~20 ml peripheral blood. Materials * Peripheral blood or buffy coat fraction * Phosphate-buffered saline (PBS), pH 7.4 (appendix 2A) * Ficoll-Hypaque (density = 1.077 g/ml) * 15- or 50-ml centrifuge tubes * Centrifuge 1. Dilute peripheral blood or the buffy coat fraction 1:1 (v/v) with PBS. Gently layer on top of a layer of Ficoll-Hypaque such that the Ficoll-Hypaque layer constitutes one-third of the total volume. Depending on the blood volume, use either 15- or 50-ml tubes. For convenience, tubes (available from Sigma) containing a special grid that separates the blood from the Ficoll can be used. 2. Centrifuge 20 min at 400 × g, at room temperature. Do not apply the brake. 3. Aspirate off the top layer (consisting of plasma and platelets), collect the interphase layer of mononuclear cells with a pipet, and transfer to a 50-ml tube. 4. Add 40 ml PBS, mix, then centrifuge 5 min at 160 × g, room temperature. Aspirate the supernatant, then resuspend the pellet in the residual buffer by tapping. 5. Add 40 ml PBS, mix, then centrifuge 5 min at 100 × g, room temperature, and aspirate the supernatant. Repeat this procedure for an additional wash. Use low-speed centrifugation in order to remove residual platelets. All procedures should be performed at room temperature. If the blood cannot be processed on the same day, leave overnight at room temperature. In experiments where Hb content is measured, special care has to be taken to avoid contamination by mature RBC that originated from the processed blood. This does not usually present a problem when normal blood is processed, but may occur with blood derived from patients, e.g., with thalassemia. The contaminating RBCs can be of patient origin and/or derived from transfused blood. In both cases, they may interfere with measurement of the Hb produced in vitro. When the interphase layer is heavily contaminated with RBC, the latter cells should be removed by osmotic hemolysis (see Support Protocol 6) prior to culturing. When only slightly contaminated, the mononuclear cells may be cultured without hemolysis; RBC tend to lyse and disappear during culture. Alternatively, they may be removed by Percoll separation at the second phase of the culture (see Support Protocol 7). [Top of Page] Basic Protocol 2: Erythroid Cell Culture Procedure The culture procedure is divided into two phases. The first is an EPO-independent phase in which peripheral blood mononuclear cells are isolated (described in Basic Protocol 1) and cultured in the presence of a combination of growth factors, but in the absence of EPO. During this phase, early erythroid-committed progenitors, called burst-forming units erythroid (BFUe), proliferate and differentiate into colony-forming unit erythroid (CFUe)–like progenitors. In the second phase, these cells, cultured in an EPO-supplemented medium, continue to proliferate and mature into orthochromatic normoblasts and enucleated erythrocytes. Materials * Complete MEM medium (see recipe) * Preselected FBS (see recipe); do not heat-inactivate * 50 mg/ml cyclosporin A (Sandoz) * Conditioned medium from 5637 human bladder carcinoma cell line (see recipe) * Mononuclear cells, washed (see Basic Protocol 1) * 10% (w/v) deionized BSA (see recipe) * 2-mercaptoethanol (Sigma) * 4 mg/ml dexamethasone sodium phosphate (Elkin-Sinn Pharmaceuticals, Cherry Hill N.J.) * Human holo-transferrin (Sigma) * Human recombinant stem cell factor (CytoLab Ltd.; http://www.cytolab.com/) * Human recombinant erythropoietin (EPO; Ortho Pharmaceutical) * 25- or 75-cm2 tissue culture flasks 1. Prepare Phase I medium by adding the following ingredients to complete MEM medium: * 10% (v/v) preselected FBS (not heat-inactivated) * 1 µg/ml cyclosporin A (add from 50 mg/ml stock) * 10% (v/v) conditioned medium from 5637 human bladder carcinoma cell line. 2a. For cells obtained from peripheral blood (10 to 20 ml): Plate the washed mononuclear cells in Phase I medium in 25-cm2 flasks at a culture volume equivalent to the original volume of blood. 2b. In the case of cells obtained from a whole blood unit (400 to 500 ml): Plate the washed mononuclear cells in Phase I medium in two 75-cm2 flasks at a culture volume of 80 to 100 ml. 3. Incubate the cultures at 37°C in a humidified, 5% CO2 incubator for 5 to 7 days. Full humidification is achieved by leaving a tray with water standing at the bottom of the incubator. 4. After the 5- to 7-day incubation in phase I, shake the cultures and harvest the nonadherent cells (do not trypsinize). 5. Centrifuge the cells 5 min at 160 × g, room temperature, and aspirate the supernatant. Fill the tube with complete MEM, centrifuge 5 min at 160 × g, room temperature, and aspirate the supernatant. Repeat for a second wash. 6. Prepare Phase II medium by adding the following ingredients to complete MEM medium: * 30% (v/v) preselected FBS (not heat-inactivated) * 1% (w/v) deionized BSA (add from 10% w/v stock) * 10–5 M 2-mercaptoethanol * 10–6 dexamethasone (add from 4 mg/ml stock) * 0.3 mg/ml human holo-transferrin * 10 ng/ml human recombinant stem cell factor * 1 U/ml human erythropoietin (EPO). 7. Plate cells in the original volume of phase II medium (as in step 2a or 2b according to the original blood volume) and incubate up to 14 days at 37°C in a humidified, 5% CO2, incubator.
Extracted molecule genomic DNA
Extraction protocol Protocol: Chromatin Immunoprecipitation coupled with Microarray Analysis (ChIP-chIP): I. Dynabead Preparation -Make PBS/BSA -For each ChIP reaction. Wash 100ul of beads 3-4 times with PBS BSA (5mg/ml) -Transfer beads to a 500ul PCR tube. -For ATRX antibody binding, Add 200ul ATRX H300, 22ul 50mg/ml BSA, 4ul ssDNA (Invitrogen 10mg/ml) -For NoAb Rxn. Add 222ul PBS BSA 4ul ssDNA -Incubate 3-4 hrs on roller at 4 oC -Wash 4 times in PBS BSA -Resuspend in 100ul PBSBSA and add 4ul (40ug) ssDNA For each preclear reaction -Take 100ul of Dynabeads -Wash 3-4 times with 1ml PBS BSA (5mg/ml freshly prepared) -Add 100ul of PBS BSA(5ug/ul) -Add 4ul ssDNA (10ug/ul) -Incubate at 4oC on roller with antibody binding reactions -Use 100ul for each preclearing reaction II. Cell cross-linking: Need 4oC PBS, Formaldehyde, EGS 1M glycine, Trypan blue, gilsons, timer 1.5ml tubes, calculator, A. Suspension Cells: 1. Make up EGS (Pierce 21565) stock fresh: 0.046g EGS in 200ul DMSO for 500mM stock 2. Chill PBS 3. Wash twice in PBS and resuspend in 20mls PBS (Important as FCS inhibits EGS) 4. Count cells 5. Use 3x107 – 5x107 cells for each ChIP reaction 6. Add EGS (it will form a white PPT which disperses afer a second) to 2mM and incubate at room temperature of 45 mins with occasional mixing. (80ul per 20mls of PBS). 7. Add formaldehyde solution to 1% for 20mins (526ul /20mls. 4 x 131.5ul) 8. Add 1/8 volume of 1M glycine to tubes to quench formaldehyde.(2.5ml glycine / 20ml PBS). 9. Spin cells at 1k for 5 min at 4oC. Make up protease inhibitors and add to lysis buffers. 10.Wash twice with 20ml 4oC PBS III. Cell sonication (keep all steps on ice): Note: Add protease inhibitors to all lysis buffers before use. If using a protease inhibitor cocktail tablet from Complete, dissolve one tablet in 2 ml water for 25X solution. Store aliquots at -20oC. 1. Resuspend each tube of cells in 10 ml of lysis buffer I. Rock at 4oC for 10 min, and then spin at 2k in a table-top centrifuge for 2 min at 4oC. 2. Resuspend each tube of cells in 5-10 ml of lysis buffer 2. Pellet nuclei in table-top centrifuge by spinning at 2.5k for 2 min at 4oC. 3. Resuspend the pellet in each tube in 3 ml lysis buffer 3. (Retain the buffer for step 6) 4. Sonicate the suspension in 15ml corning blue top tubes. 2mls per tube. Use the Bioruptor at high power for 20mins (4x5mins). After each 5 minute pulse, replenish the ice. 5. Add 1/10 volume of 10% Triton X-100. Transfer to 1.5 ml centrifuge tubes. Spin out debris at 14k for 10 min at 4oC. The lysate contains the fragmented DNA. 6. Save pellet for western analysis and take 50ul lysate for western analysis 7. Transfer lysate to 15 ml conical tube, adjusting the volume to 3 ml using lysis buffer 3. 8. Save 50µl of cell lysate from each sample as input. Save 50ul to assess sonication efficiency. IV. Chromatin immunoprecipitation: -Add 100ul of the preclearing beads from step I to the 3mls of chromatin lysate -Preclear on roller at 4 oC for 1 hour -Precipitate beads from chromatin using magnet and remove chromatin to new 3ml tubes -Add the washed antibody bound beads from step 1 -Incubate overnight on roller at 4 oC V. Washing, eluting, and reverse cross-linking: Note: Carry steps 1-3 in the cold room and all wash solution must be ice cold. 1. Transfer lysates from the 3 ml tubes to a 1.5ml tube. 2. Wash the IPs 5 times with 1 ml wash buffer (be sure to add protease inhibitors to the wash buffer). Use the magnetic stand to precipitate the beads. 3. Wash once with 1 ml cold PBS. Aspirate residual PBS. 4. Add 250µl of elution buffer. Elute on a roller at room temperature for 15 minutes. 5. Add 250ul more of elution buffer to beads, vortex, precipitate and combine with previous round of elution. 6. Take 50ul of eluate for western blot analysis 7. PPT beads and remove eluate to a new tube. Add 20ul 5M NaCl and incubate at 65oC for 4 hours. Thaw input, make up to 500ul with elution buffer, add 20ul 5M NaCl and incubate for 4hrs at 65oC. VI. RNase, Proteinase K, Phenol/Chloroform, EtOH PPT: Remove beads. Then add 20ul of 1M Tris pH6.5, 10ul 0.5M EDTA, 2ul 10mg/mg Proteinase K. Incubate for 1hrs at 45°C. Phenol chloroform and EtOH PPT with 20ug of carrier glycogen. Resuspend in 21ul sigma water. Use low volume to give the option of labelling for ChIP on Chip. For real time PCR, use 1/12 ul per reaction Solutions PBS BSA 5mg/ml For 50mls: 5mls 10XPBS, 250mg BSA Fraction V, Filter sterilise. Make up fresh every time. Lysis Buffer 1 (100 ml): 100mM HEPES 140mM NaCl 1mM EDTA 10% Glycerol 0.5% NP40 0.25% Triton X Lysis Buffer 2 (100 ml): 200mM NaCl 1mM EDTA 0.5mM EGTA 10mM Tris pH 8 Lysis Buffer 3 (100 ml): 1mM EDTA 0.5mM EGTA 1mMTris-HCl pH8 100mM NaCl 0.1% Na DOC 0.5% N-lauroyl Sarcosine Wash buffer (RIPA Buffer, 100 ml): 50mM Hepes, pH 7.6 1mM EDTA 0.7% Na DOC 1% NP40 500mM LiCl
Label Cy5
Label protocol Random Labelling DNA for array CGH Equipment and reagents • BioPrime Labelling Kit (Invitrogen 18094-011) • 10X dNTP mix (Amersham 27-2035-01) • 1mM Cy3-dCTP (Amersham PA53021) • 1mM Cy5-dCTP (Amersham PA55021) • HPLC water BDH 152736D • Micro-spin G50 columns (Amersham, 275330-01) Method 1. Random label 450 ng of DNA in a final reaction volume of 150 μl by mixing the DNA with 60 μl 2.5X Random Primers Solution (from BioPrime Labelling Kit) and make up to 130 μl with HPLC water. Make 1 reaction for ChIP DNA and 1 reaction for input DNA. 2. Denature DNA in a heat block for 10 min at 100°C, and immediately cool on ice. 3. The following reagents are added on ice: 15 μl 10X dNTP mix (so prepared: dNTPs Amersham 27-2035-01 4x25umol C 1μl , A 2 μl, G 2 μl , T 2 μl , HPLC water 93 μl) 1.5 μl Cy3 (for ChIP DNA) or Cy5 (for input DNA) labelled dCTP (1 mM) from Amersham PA53021 and PA55021 3 μl Klenow Fragment from Biopreime Labelling Kit Mix gently but thoroughly. 4. Incubate the reaction @ 37°C overnight in darkness and then stop it by adding 15 μl of stop buffer (from BioPrime Labelling kit). Removal of Labelled Nucleotides from DNA Labelling Reactions (The recommended volume for application to a single column is 50 μl, you therefore have to use 3 columns per 150 µl labelling reaction) Column Preparation 1. Resuspend the resin in the columns by gentle vortexing. 2. Loosen the cap one-fourth turn and snap off the bottom closure. 3. Place the columns in a 1.5 ml screw-cap microcentrifuge tube for support. Alternatively, cut the cap from a flip-top tube and use this tube for support. 4. Pre-spin the columns for 1 minute at 2700 rpm. DO NOT pulse as this will override the variable speed setting. Wash columns applying 50 mcl of HPLC water to the resin and spin 1 min at 2700 rpm. Use columns immediately after preparation to avoid the resin drying out. Sample Application 1. Place the columns in a new 1.5 ml tube and slowly apply 50 μl to the centre of the angled surface of the compacted resin bed of each of the columns, being careful not to disturb the resin bed. Careful application of the sample to the centre of the bed is essential for good separation. Do not allow any of the samples to flow around the sides of the bed. 2. Spin the columns for 2 minutes at 2700 rpm. The purified samples are collected in the bottom of the support tube. 3. Discard the columns and retain the flow-through samples. 4. Combine the three samples and run 5 μl on a 1% agarose gel
 
 
Hybridization protocol Hybridisation of labelled ChIP DNA onto Microarrays 1. Prepare in 2μl eppendorf tube the hybridisation DNA mixtures for precipitation as follows: a. 160 μl Cy3 labelled DNA b. 160 μl Cy labelled DNA c. 135 μl Human Cot-1 DNA (Invitrogen 15279-011) d. 55 μl 3M NaAc ph 5.2 e. 6 μl yeast tRNA (Invitrogen 15401-011) f. 1000 μl 100% EtOH 2. Mix the tube gently, keep in dark and precipitate for 30 min – 1 h at –70^C 3. Pre heat hybridisation buffer in a 65^C heat block Hybridisation buffer (50 ml): 20X SSC 5 ml Deionised formamide 25 ml 100mM Tris-Hcl ph7.4 5 ml Dextran sulphate 2.5 g Tween 20 50 mcl HPLC water 15 ml 4. Spin the precipitated DNA for 20 min at 14000 rpm at 4^C 5. Wash the pellet in EtOH 70% 500 mcl and spin 5 min at 14000 rpm at 4^C 6. Leave the pellet drying 7. Resuspend pellet in 120-150 μl of hybridisation buffer (dependant on chamber size) and leave dissolving on 65^C heat block for 1+ hour 8. When the pellet is fully dissolved denature for 10 min at 100^C in darkness 9. Snap freeze on ice, spin down and leave on ice until loading in the liquid station.
Scan protocol Slides were scanned with a Perkin Elmer Scanarray scanner using scanarray express software.
Data processing The Easy Quant (Perkin Elmer, Scan Array) analysis program with 'Adaptive circle' quantitation method with lowess normalization was used. log2 ChIP / Input ratios reported
 
Submission date Jun 04, 2010
Last update date Nov 02, 2010
Contact name Richard J Gibbons
E-mail(s) richard.gibbons@imm.ox.ac.uk
Phone +441865222632
Fax +441865222500
URL http://www.imm.ox.ac.uk/wimm-research/molhaem/richard-gibbons
Organization name Weatherall Institute of Molecular Medicine
Department MRC Molecular Haematology Unit
Lab Gibbons
Street address John Radcliffe Hospital,
City Oxford
State/province Oxon
ZIP/Postal code OX3 9DU
Country United Kingdom
 
Platform ID GPL10485
Series (1)
GSE22162 A SNF2 protein targets variable copy number repeats and thereby influences allele-specific expression

Data table header descriptions
ID_REF
VALUE lowess normalized log2 ChIP/Input ratio

Data table
ID_REF VALUE
1 1.89917563
2 1.646162657
3 0.97819563
4 0.678071905
5 0.718087584
6 0.739848103
7 0.66448284
8 0.521050737
9 0.867896464
10 0.09085343
11 -0.104697379
12 0.056583528
13 0.084064265
14 -0.160040413
15 -0.074000581
16 0.384049807
17
18
19
20 0.333423734

Total number of rows: 512

Table truncated, full table size 5 Kbytes.




Supplementary file Size Download File type/resource
GSM551131_ATRX29-1-7.txt.gz 521.3 Kb (ftp)(http) TXT
Processed data included within Sample table
Raw data provided as supplementary file

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