cell line: human embryonic stem (hES) cell line NSCB# 9016, CGH# 239
Extracted molecule
genomic DNA
Extraction protocol
1. Determine When to Harvest hES cells for DNA Extraction A) At least 1x10^6 hES cells should be harvested for DNA extraction, depending on the quantity required. B) Yields of genomic DNA will vary from sample to sample depending on the amount and type of cells processed. In addition, the quality of starting material will affect DNA yield. C) Estimated yield for hES cells: 5pg of DNA per cell (5μg DNA per 10^6 cells). One well of a 6-well plate will normally have between 1-4x106 hES cells by day 5 after passage. 2. Trypsinize the Cells A) The following steps should be performed in a sterile biosafety cabinet while wearing personal protective equipment. B) Add 1.5ml of 1X trypsin-EDTA working solution (pre-warmed to 37oC) to each well of a 6- well plate. C) Return the plate to the 37C incubator for 2-3 minutes. D) Remove the plate from the incubator and observe cell detachment with an inverted phase contrast microscope. Proceed to next step when colony edges begin to lift off the plate. E) Using a sterile glass serological pipet, gently pipet up and down to detach the cells and break up the cell clumps. F) Pool the detached cells in a 15ml centrifuge tube containing 5ml of trypsin stop solution within 5-7 minutes of adding the trypsin-EDTA solution. G) Gently pipet the solution up and down to mix thoroughly. H) Centrifuge the tube for 5 minutes at 1000 RPM. 3. RNA Digestion A) Aspirate the supernatant and gently flick the tube until the pellet is resuspended. B) Slowly add 200μl PBS to the cells while tapping gently on the tube to ensure good mixing. C) Add 4μl RNase A (100mg/ml) and incubate at room temperature (RT) for 2 minutes. 4. Protein Digestion A) Add 20μl Proteinase K and 200μl Buffer AL to the sample, mixing thoroughly by vortexing. It is important that the solution is immediately and thoroughly mixed by vortexing and pipetting to yield a homogenous solution. NOTE: Do not add proteinase K directly to buffer AL. B) Incubate the cell solution in a 70°C waterbath for 10 minutes. 5. DNA Precipitation and Separation of Cellular Components A) Add 200μl ethanol (96-100%) to the sample, and mix thoroughly by vortexing. B) A white precipitate may form on addition of ethanol. This precipitate does not interfere with the DNeasy procedure. C) Pipet the mixture (including all of the precipitate) into the DNeasy Mini spin column placed in a 2ml collection tube. Centrifuge at ≥6000 x g (8000 rpm) for 1 minute. Discard flowthrough and collection tube. NOTE: In order to obtain optimum DNA yield and quality, it is important not to overload the spin column. The maximum number of hES cells that should be used in a DNA extraction is 5x106 (normally, 2 wells of a 6-well plate). For larger amounts of starting material use multiple spin columns. 6. Wash the DNA A) Place the DNeasy spin column in a new collection tube and add 500μl of Buffer AW1. B) Centrifuge at ≥6000 x g (8000rpm) for 1 minute. Discard flow-through and collection tube. C) Place the DNeasy spin column in a new collection tube and add 500μl of buffer AW2. D) Centrifuge at 20,000 x g (14,000 rpm) for 3 minutes to dry the DNeasy membrane. Discard flow-through and collection tube. NOTE: It is important to dry the membrane of the DNeasy Mini spin column, since residual ethanol may interfere with subsequent reactions. This centrifugation step ensures that no residual ethanol will be carried over during the following elution. E) Following the centrifugation step, remove the DNeasy Mini spin column carefully so the column does not come into contact with the flow-through, since this will result in carryover of ethanol. If carryover of ethanol occurs, place the spin column in a new collection tube and centrifuge again for 1 minute at 20,000 x g (14,000 rpm). Again discard the flow-through and collection tube. 7. Elution A) Place the DNeasy Mini spin column in a clean 1.5ml or 2ml microcentrifuge tube and pipet 50-200μl Buffer AE directly onto the DNeasy membrane. Incubate the column at room temperature for 1 minute and then centrifuge for 1 minute at ≥6000 x g (8000rpm) to elute. NOTE: Elution with less than 200μl of Buffer AE increases the final DNA concentration in the elute, but also decreases the overall DNA yield. B) Repeat elution step as described in 7 A). NOTE: A new microcentrifuge tube can be used for the second elution step to prevent dilution of the first elute. Alternatively, to combine the two elutes, the microcentrifuge tube from step 4.7.1 can be reused for the second elution step. NOTE: More than 200μl should not be eluted into a 1.5 ml microcentrifuge tube because the DNeasy spin column will come into contact with the elute.
Label
Cy3
Label protocol
7.7.1. Cy3/Cy5 Labeling 7.7.1.1. Verify that the DNA concentration is between 25 and 30ng/ul for all test and reference samples. 7.7.1.2. Thaw the Cy3/Cy5 random nonamers on ice, protected from light. 7.7.1.3. Add 1µg of sonicated test DNA to 40 µl Cy3 dye. Bring to volume in 80 µl VWR water. Repeat for all test samples to be labeled. 7.7.1.4. Add 1 µg of sonicated reference DNA to 40 µl Cy5 dye. Bring to volume in 80 µl VWR water. Repeat for all reference samples to be labeled. 7.7.1.5. Vortex and quickly centrifuge test and reference DNA samples. 7.7.1.6. Incubate samples for 10 minutes at 98ºC protecting from light. 7.7.1.7. Rapidly transfer samples to an ice-water bath for 5 minutes protecting from light. 7.7.1.8. Prepare 20µl of the following aha-dNTP/Klenow Master Mix for each test and reference sample. 7.7.1.8.1. aha-dNTP/Klenow Master Mix per Sample 7.7.1.8.1.1. Thaw all reagents and aha-dNTP/Klenow master mix on ice. 7.7.1.8.1.2. Add 9µl VWR water to 0.2ml PCR tube. 7.7.1.8.1.3. Add 10µl 50X aha-dNTP Mix. 7.7.1.8.1.4. Add 1µl Klenow (50U/µl). DO NOT vortex. Total amount of solution is 20µl. 7.7.1.9. Add 20μl of the aha-dNTP/Klenow Master Mix to each of the cooled samples. Keep on ice. 7.7.1.10. Pipette to mix (15 times). DO NOT vortex. 7.7.1.11. Briefly centrifuge samples using a tabletop centrifuge. 7.7.1.12. Incubate the samples for 2 hours 15 minutes at 37°C in thermocycler protected from light. 7.7.1.13. Remove samples from thermocycler, keep at room temperature. Heat thermocycler to 98ºC. Place samples back into thermocycler and incubate for 10 minutes at 98ºC. 7.7.1.14. Rapidly transfer samples to an ice-water bath for 5 minutes protecting samples from light. 7.7.1.15. Add 1 µl of Klenow (50U/µl) to each sample. 7.7.1.16. Pipette to mix (15 times). DO NOT vortex. 7.7.1.17. Briefly centrifuge samples using a tabletop centrifuge. 7.7.1.18. Place samples in thermocycler at 37ºC for overnight amplification. 7.7.2. First DNA Precipitation 7.7.2.1. Set Roche Nimblegen Hybridization System 12 to 48ºC if proceeding to hybridization in the same day to allow for at least 3 hours of stabilization. 7.7.2.2. Cool centrifuge to 4ºC. 7.7.2.3. Add 10µl of 0.5M EDTA to each sample to stop the overnight amplification reaction. 7.7.2.4. Add 11.5 µl of 5M NaCl to each sample, then vortex for 10 seconds. 7.7.2.5. Briefly centrifuge samples using a tabletop centrifuge. 7.7.2.6. Transfer each sample into a 1.5ml tube containing 200 µl of isopropanol. 7.7.2.7. Vortex samples for 20 seconds. 7.7.2.8. Incubate at room temperature for 10 minutes, protected from light. 7.7.2.9. Centrifuge at 13,000 rpm 4ºC for 10 minutes. 7.7.2.10. Aspirate supernatant. 7.7.2.11. Rinse pellet with 500µl of 80% ice-cold ethanol. As you rinse, gently wash pellet from tube wall. 7.7.2.12. Centrifuge at 13,000 rpm 4ºC for 2 minutes. 7.7.2.13. Aspirate supernatant. 7.7.2.14. Dry contents in a SpeedVac with tube lids open on ‘low’ heat until dry (5-15 minutes), protected from light. Close lids when dried. 7.7.2.15. Proceed to Alexa Fluor Labeling or store labeled samples at -20°C, protected from light. 7.7.2.15.1. Frozen samples are stable for up to 2 weeks at -20ºC. 7.7.3. Alexa Fluor Labeling 7.7.3.1. Thaw one vial of Alexa Fluor 555 dye per Cy3-labeled test DNA sample on ice. Keep protected from light. 7.7.3.2. Thaw one vial of Alexa Fluor 647 dye per Cy5-labeled reference DNA sample on ice. Keep protected from light. 7.7.3.3. Thaw Alexa Fluor Labeling Buffer on ice. 7.7.3.4. Add 5 µl VWR water to each DNA sample. 7.7.3.5. Place samples in thermocycler at 42ºC for 5 minutes. 7.7.3.6. Add 3 µl Labeling Buffer to each DNA sample. 7.7.3.7. Vortex and quickly centrifuge DNA samples. 7.7.3.8. Add 2 µl DMSO to one of the vials of Alexa Fluor dye (either 555 or 647). 7.7.3.9. Vortex for 10 seconds to dissolve Alexa Fluor dye. 7.7.3.10. Immediately add 8 µl of corresponding DNA to the dissolved dye. 7.7.3.11. Pipette up and down to further dissolve dye pellet, replace lid and vortex briefly to mix. 7.7.3.12. Incubate at room temperature, protected from light for 1 hour. 7.7.3.13. Repeat steps 7.7.3.8.-7.7.3.12 for each Cy-labeled DNA to be labeled with Alexa Fluor dyes. 7.7.4. Second DNA Precipitation 7.7.4.1. Add 100 µl VWR water to each labeled DNA sample. 7.7.4.2. Add 11.5 µl of 5M NaCl to each sample. 7.7.4.3. Vortex for 10 seconds. 7.7.4.4. Briefly centrifuge samples using a tabletop centrifuge. 7.7.4.5. Transfer each sample into 1.5ml tubes containing 200 µl each of isopropanol. 7.7.4.6. Vortex samples for 20 seconds. 7.7.4.7. Incubate at room temperature for 10 minutes, protected from light. 7.7.4.8. Centrifuge at 13,000 rpm 4ºC for 10 minutes. 7.7.4.9. Aspirate supernatant. 7.7.4.10. Add 500µl of 80% ice-cold ethanol to the pellet and gently wash pellet from tube wall by pipetting. 7.7.4.11. Centrifuge at 13,000 rpm 4ºC for 2 minutes. 7.7.4.12. Aspirate supernatant. 7.7.4.13. Dry contents in a SpeedVac with tube lids open on ‘low’ heat until dry (5-15 minutes), protected from light. Close lids when dried. 7.7.4.14. Proceed to ‘Specific Activity Calculation’ or store labeled samples at -20°C, protected from light. Frozen samples are stable for up to 2 weeks at -20ºC.
cell line: human embryonic stem (hES) cell line H9 [reference]
Extracted molecule
genomic DNA
Extraction protocol
1. Determine When to Harvest hES cells for DNA Extraction A) At least 1x10^6 hES cells should be harvested for DNA extraction, depending on the quantity required. B) Yields of genomic DNA will vary from sample to sample depending on the amount and type of cells processed. In addition, the quality of starting material will affect DNA yield. C) Estimated yield for hES cells: 5pg of DNA per cell (5μg DNA per 10^6 cells). One well of a 6-well plate will normally have between 1-4x106 hES cells by day 5 after passage. 2. Trypsinize the Cells A) The following steps should be performed in a sterile biosafety cabinet while wearing personal protective equipment. B) Add 1.5ml of 1X trypsin-EDTA working solution (pre-warmed to 37oC) to each well of a 6- well plate. C) Return the plate to the 37C incubator for 2-3 minutes. D) Remove the plate from the incubator and observe cell detachment with an inverted phase contrast microscope. Proceed to next step when colony edges begin to lift off the plate. E) Using a sterile glass serological pipet, gently pipet up and down to detach the cells and break up the cell clumps. F) Pool the detached cells in a 15ml centrifuge tube containing 5ml of trypsin stop solution within 5-7 minutes of adding the trypsin-EDTA solution. G) Gently pipet the solution up and down to mix thoroughly. H) Centrifuge the tube for 5 minutes at 1000 RPM. 3. RNA Digestion A) Aspirate the supernatant and gently flick the tube until the pellet is resuspended. B) Slowly add 200μl PBS to the cells while tapping gently on the tube to ensure good mixing. C) Add 4μl RNase A (100mg/ml) and incubate at room temperature (RT) for 2 minutes. 4. Protein Digestion A) Add 20μl Proteinase K and 200μl Buffer AL to the sample, mixing thoroughly by vortexing. It is important that the solution is immediately and thoroughly mixed by vortexing and pipetting to yield a homogenous solution. NOTE: Do not add proteinase K directly to buffer AL. B) Incubate the cell solution in a 70°C waterbath for 10 minutes. 5. DNA Precipitation and Separation of Cellular Components A) Add 200μl ethanol (96-100%) to the sample, and mix thoroughly by vortexing. B) A white precipitate may form on addition of ethanol. This precipitate does not interfere with the DNeasy procedure. C) Pipet the mixture (including all of the precipitate) into the DNeasy Mini spin column placed in a 2ml collection tube. Centrifuge at ≥6000 x g (8000 rpm) for 1 minute. Discard flowthrough and collection tube. NOTE: In order to obtain optimum DNA yield and quality, it is important not to overload the spin column. The maximum number of hES cells that should be used in a DNA extraction is 5x106 (normally, 2 wells of a 6-well plate). For larger amounts of starting material use multiple spin columns. 6. Wash the DNA A) Place the DNeasy spin column in a new collection tube and add 500μl of Buffer AW1. B) Centrifuge at ≥6000 x g (8000rpm) for 1 minute. Discard flow-through and collection tube. C) Place the DNeasy spin column in a new collection tube and add 500μl of buffer AW2. D) Centrifuge at 20,000 x g (14,000 rpm) for 3 minutes to dry the DNeasy membrane. Discard flow-through and collection tube. NOTE: It is important to dry the membrane of the DNeasy Mini spin column, since residual ethanol may interfere with subsequent reactions. This centrifugation step ensures that no residual ethanol will be carried over during the following elution. E) Following the centrifugation step, remove the DNeasy Mini spin column carefully so the column does not come into contact with the flow-through, since this will result in carryover of ethanol. If carryover of ethanol occurs, place the spin column in a new collection tube and centrifuge again for 1 minute at 20,000 x g (14,000 rpm). Again discard the flow-through and collection tube. 7. Elution A) Place the DNeasy Mini spin column in a clean 1.5ml or 2ml microcentrifuge tube and pipet 50-200μl Buffer AE directly onto the DNeasy membrane. Incubate the column at room temperature for 1 minute and then centrifuge for 1 minute at ≥6000 x g (8000rpm) to elute. NOTE: Elution with less than 200μl of Buffer AE increases the final DNA concentration in the elute, but also decreases the overall DNA yield. B) Repeat elution step as described in 7 A). NOTE: A new microcentrifuge tube can be used for the second elution step to prevent dilution of the first elute. Alternatively, to combine the two elutes, the microcentrifuge tube from step 4.7.1 can be reused for the second elution step. NOTE: More than 200μl should not be eluted into a 1.5 ml microcentrifuge tube because the DNeasy spin column will come into contact with the elute.
Label
Cy5
Label protocol
7.7.1. Cy3/Cy5 Labeling 7.7.1.1. Verify that the DNA concentration is between 25 and 30ng/ul for all test and reference samples. 7.7.1.2. Thaw the Cy3/Cy5 random nonamers on ice, protected from light. 7.7.1.3. Add 1µg of sonicated test DNA to 40 µl Cy3 dye. Bring to volume in 80 µl VWR water. Repeat for all test samples to be labeled. 7.7.1.4. Add 1 µg of sonicated reference DNA to 40 µl Cy5 dye. Bring to volume in 80 µl VWR water. Repeat for all reference samples to be labeled. 7.7.1.5. Vortex and quickly centrifuge test and reference DNA samples. 7.7.1.6. Incubate samples for 10 minutes at 98ºC protecting from light. 7.7.1.7. Rapidly transfer samples to an ice-water bath for 5 minutes protecting from light. 7.7.1.8. Prepare 20µl of the following aha-dNTP/Klenow Master Mix for each test and reference sample. 7.7.1.8.1. aha-dNTP/Klenow Master Mix per Sample 7.7.1.8.1.1. Thaw all reagents and aha-dNTP/Klenow master mix on ice. 7.7.1.8.1.2. Add 9µl VWR water to 0.2ml PCR tube. 7.7.1.8.1.3. Add 10µl 50X aha-dNTP Mix. 7.7.1.8.1.4. Add 1µl Klenow (50U/µl). DO NOT vortex. Total amount of solution is 20µl. 7.7.1.9. Add 20μl of the aha-dNTP/Klenow Master Mix to each of the cooled samples. Keep on ice. 7.7.1.10. Pipette to mix (15 times). DO NOT vortex. 7.7.1.11. Briefly centrifuge samples using a tabletop centrifuge. 7.7.1.12. Incubate the samples for 2 hours 15 minutes at 37°C in thermocycler protected from light. 7.7.1.13. Remove samples from thermocycler, keep at room temperature. Heat thermocycler to 98ºC. Place samples back into thermocycler and incubate for 10 minutes at 98ºC. 7.7.1.14. Rapidly transfer samples to an ice-water bath for 5 minutes protecting samples from light. 7.7.1.15. Add 1 µl of Klenow (50U/µl) to each sample. 7.7.1.16. Pipette to mix (15 times). DO NOT vortex. 7.7.1.17. Briefly centrifuge samples using a tabletop centrifuge. 7.7.1.18. Place samples in thermocycler at 37ºC for overnight amplification. 7.7.2. First DNA Precipitation 7.7.2.1. Set Roche Nimblegen Hybridization System 12 to 48ºC if proceeding to hybridization in the same day to allow for at least 3 hours of stabilization. 7.7.2.2. Cool centrifuge to 4ºC. 7.7.2.3. Add 10µl of 0.5M EDTA to each sample to stop the overnight amplification reaction. 7.7.2.4. Add 11.5 µl of 5M NaCl to each sample, then vortex for 10 seconds. 7.7.2.5. Briefly centrifuge samples using a tabletop centrifuge. 7.7.2.6. Transfer each sample into a 1.5ml tube containing 200 µl of isopropanol. 7.7.2.7. Vortex samples for 20 seconds. 7.7.2.8. Incubate at room temperature for 10 minutes, protected from light. 7.7.2.9. Centrifuge at 13,000 rpm 4ºC for 10 minutes. 7.7.2.10. Aspirate supernatant. 7.7.2.11. Rinse pellet with 500µl of 80% ice-cold ethanol. As you rinse, gently wash pellet from tube wall. 7.7.2.12. Centrifuge at 13,000 rpm 4ºC for 2 minutes. 7.7.2.13. Aspirate supernatant. 7.7.2.14. Dry contents in a SpeedVac with tube lids open on ‘low’ heat until dry (5-15 minutes), protected from light. Close lids when dried. 7.7.2.15. Proceed to Alexa Fluor Labeling or store labeled samples at -20°C, protected from light. 7.7.2.15.1. Frozen samples are stable for up to 2 weeks at -20ºC. 7.7.3. Alexa Fluor Labeling 7.7.3.1. Thaw one vial of Alexa Fluor 555 dye per Cy3-labeled test DNA sample on ice. Keep protected from light. 7.7.3.2. Thaw one vial of Alexa Fluor 647 dye per Cy5-labeled reference DNA sample on ice. Keep protected from light. 7.7.3.3. Thaw Alexa Fluor Labeling Buffer on ice. 7.7.3.4. Add 5 µl VWR water to each DNA sample. 7.7.3.5. Place samples in thermocycler at 42ºC for 5 minutes. 7.7.3.6. Add 3 µl Labeling Buffer to each DNA sample. 7.7.3.7. Vortex and quickly centrifuge DNA samples. 7.7.3.8. Add 2 µl DMSO to one of the vials of Alexa Fluor dye (either 555 or 647). 7.7.3.9. Vortex for 10 seconds to dissolve Alexa Fluor dye. 7.7.3.10. Immediately add 8 µl of corresponding DNA to the dissolved dye. 7.7.3.11. Pipette up and down to further dissolve dye pellet, replace lid and vortex briefly to mix. 7.7.3.12. Incubate at room temperature, protected from light for 1 hour. 7.7.3.13. Repeat steps 7.7.3.8.-7.7.3.12 for each Cy-labeled DNA to be labeled with Alexa Fluor dyes. 7.7.4. Second DNA Precipitation 7.7.4.1. Add 100 µl VWR water to each labeled DNA sample. 7.7.4.2. Add 11.5 µl of 5M NaCl to each sample. 7.7.4.3. Vortex for 10 seconds. 7.7.4.4. Briefly centrifuge samples using a tabletop centrifuge. 7.7.4.5. Transfer each sample into 1.5ml tubes containing 200 µl each of isopropanol. 7.7.4.6. Vortex samples for 20 seconds. 7.7.4.7. Incubate at room temperature for 10 minutes, protected from light. 7.7.4.8. Centrifuge at 13,000 rpm 4ºC for 10 minutes. 7.7.4.9. Aspirate supernatant. 7.7.4.10. Add 500µl of 80% ice-cold ethanol to the pellet and gently wash pellet from tube wall by pipetting. 7.7.4.11. Centrifuge at 13,000 rpm 4ºC for 2 minutes. 7.7.4.12. Aspirate supernatant. 7.7.4.13. Dry contents in a SpeedVac with tube lids open on ‘low’ heat until dry (5-15 minutes), protected from light. Close lids when dried. 7.7.4.14. Proceed to ‘Specific Activity Calculation’ or store labeled samples at -20°C, protected from light. Frozen samples are stable for up to 2 weeks at -20ºC.
Hybridization protocol
7.9.1. Set the Roche NimbleGen Hybridization System 12 to 48°C. With the cover closed, allow at least 3 hours for the temperature to stabilize. 7.9.2. Hybridization Solution Master Mix per Chip 7.9.2.1. Thaw Alignment Oligo on ice, protected from light. 7.9.2.2. Add 11.8µl 2X Hybridization Buffer to 0.2ml PCR tube. 7.9.2.3. Add 4.7µl Hybridization Component A. 7.9.2.4. Add 0.5µl Alignment Oligo. Total amount of solution is 17.0µl. 7.9.2.5. Vortex to mix. 7.9.2.6. Briefly centrifuge samples using a tabletop centrifuge and keep on ice, protected from light. 7.9.3. Combine Test and Reference DNA Samples 7.9.3.1. Quantify each sample using the NanoDrop as described in SOP-SS-001 7.9.3.2. Combine 6µg each of test and reference labeled DNA in a PCR tube and bring to total volume of 5µl with VWR water. The combined volume of the test and reference samples must not exceed 5.0µl. Each of the labeled samples must fall within the concentration range of 2400-3400/ml. 7.9.3.2.1. If the concentration of the labeled sample is low, dehydrate the labeled sample in the SpeedVac at high heat for 3 minutes with tube lids open. Monitor the volume of sample to prevent complete dehydration. 7.9.3.2.2. If the concentration is still too low, reprecipitate starting from the second DNA precipitation (do NOT add 11.5ul of 5M NaCl) and rehydrate the pellet in a smaller volume of VWR water. 7.9.3.2.3. If the concentration of either sample is greater than 3400 µg/ml, dilute with VWR water to achieve the acceptable concentration range. 7.9.3.3. Add 13µl Hybridization Solution Master Mix to combined labeled DNA. 7.9.3.4. Vortex the samples for 20 seconds and quickly centrifuge in tabletop centrifuge. Keep samples protected from light. 7.9.3.5. Place samples in 95ºC thermocycler for 5 minutes. 7.9.3.6. Briefly vortex and centrifuge samples. 7.9.3.7. Quickly transfer the 0.2ml PCR tubes to the Roche NimbleGen Hybridization System 12 sample block (pre-heated to 48°C) for at least 5 minutes before sample loading. 7.9.4. Prepare X1 Mixer 7.9.4.1. Prepare X1 mixer according to NimbleGen™ Arrays User Guide (CGH Analysis v5.0). 7.9.5. Load Sample 7.9.5.1. Quickly transfer labeled DNA sample from PCR tube in 48ºC sample block to a 2.0 ml tube. 7.9.5.2. Dispense 18µl of sample into the bottom X1 mixer hole using a Gilson Microman M100 pipette. 7.9.5.3. Dry any excess sample from X1 mixer holes using a Kimwipe. 7.9.5.4. Seal top and bottom X1 mixer holes with mixer port seals. 7.9.5.5. Close the bay clamp. 7.9.5.6. Dampen humid cover with Millipore water and place over bays. Humid covers must be kept damp throughout hybridization. 7.9.5.7. Close the Roche NimbleGen Hybridization System 12 lid. 7.9.5.8. Turn on the Mixing Panel on the Hybridization System, set the mix mode to B, and press the mix button to start mixing. 7.9.5.9. Confirm that the Hybridization System recognizes the slide in each occupied bay by indicator light turning green. 7.9.5.10. Hybridize the sample to the array(s) for 40-42 hours at 48°C. 7.10. Sample Storage 7.10.1. Label the remaining labeled DNA according to the following format: 7.10.2. Store labeled DNA samples at -20C in “CGH Labeled Samples” box for up to two weeks. 7.11. Array Washing 7.11.1. Turn on GenePix 4000B to warm up lasers prior to beginning wash procedure. 7.11.2. Disassemble and wash arrays according to NimbleGen™ Arrays User Guide (CGH Analysis v5.0) with the following exceptions: 7.11.2.1. Heat both Wash Buffer I solutions to 42ºC. 7.11.2.2. Agitate array in Wash Buffer I for 3 minutes. 7.11.2.3. Follow 1 minute agitation in Wash Buffer 2 with a 2 minute incubation. 7.11.3. Perform washes protecting arrays from light and ozone. 7.11.4. Place array in corresponding case and wrap case in aluminum foil. 7.11.4.1. Label with CGH number. 7.11.5. Scan arrays immediately after washing (ref: SOP-CH-309). 7.12. Array Storage 7.12.1. Store scanned arrays in the dessicator.
Scan protocol
NimbleGen scan protocol
Description
This CGH experiment is designed to detect deletion, insertation, amplication, etc. in the sample relative to the reference.
Data processing
1. Image extraction: NimbleScan provided by NimbleGen is used to extract signal data from image files (*.TFF). Signals from the scan for Cy3 (green channel) are saved in a file named as chipID_532.pair while signals from the scan for Cy5 (red channel) are saved in a file named as chipID_635.pair.
2. Normalization: Signals from Cy3/Cy5 channels are transformed by logarithms to base 2 and normalized with loess or qspline (Workman et al., 2002) algorithms by R to resolve the systematic errors and bias introduced by the microarray experimental platform. The log ratios of the signals are obtained by subtracting the log transformed signals from one channel by the corresponding ones from another channel.
3. Data smoothing and break-points identification: Ratios of the normalized data are plotted by the corresponding genome locations. The data are smoothed by an adaptive weights smoothing algorithm (GLAD R-package, Wang et al., 2005), break-points are identified by a binary segmentation algorithm (DNAcopy R-package, Olshen et al., 2004) or by the segmentation algorithm available within Nimbelgen’s Nimblescan software.
4. Data visualization: CGH results can be saved as SignalMap GFF files and can be visualized by SignalMap provided by NimbleGen. CGH results can also be visualized by other software, such as aCGHViewer (Shankar et al., 2006), CHARMView (Myers et al., 2005), etc. However, different software requires different input format.
