cell type: human embryonic stem (hES) cell line: SOM02V4Y
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
1. Dry the sample in the Speed Vac set at 30°C. 2. Resuspend the sample in 4.5μl 0.1 M Na2CO3, pH 9.0. 3. Resuspend the Cy3 NHS ester in 4.5μl DMSO 4. Add Cy3 NHS ester to sample 5. Incubate at 22°C in a heat block for 1 hour. Protect the heat block and sample from light. 6. Purify the sample using a Qiaquick PCR purification Kit: A) Add 35μl 100 mM NaOAc, pH 5.2 to the sample. B) Add 250μl PB buffer to the sample and load the sample on the column. C) Spin at 13.000 rpm for 1 minute and remove the flow through. D) Add 750μl PE buffer to the column. E) Spin at 13.000 rpm for 1 minute and remove the flow through. F) Spin at 13.000 rpm for 1 minute. G) Transfer the column to new tube. H) Add 30μl H20 and incubate for 1 minute on the column. I) Elute once by centrifuging at 13.000 rpm for 1 minute.
cell type: human embryonic stem (hES) cell line: SOM02V52
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
1. Dry the sample in the Speed Vac set at 30°C. 2. Resuspend the sample in 4.5μl 0.1 M Na2CO3, pH 9.0. 3. Resuspend the Cy3 NHS ester in 4.5μl DMSO 4. Add Cy3 NHS ester to sample 5. Incubate at 22°C in a heat block for 1 hour. Protect the heat block and sample from light. 6. Purify the sample using a Qiaquick PCR purification Kit: A) Add 35μl 100 mM NaOAc, pH 5.2 to the sample. B) Add 250μl PB buffer to the sample and load the sample on the column. C) Spin at 13.000 rpm for 1 minute and remove the flow through. D) Add 750μl PE buffer to the column. E) Spin at 13.000 rpm for 1 minute and remove the flow through. F) Spin at 13.000 rpm for 1 minute. G) Transfer the column to new tube. H) Add 30μl H20 and incubate for 1 minute on the column. I) Elute once by centrifuging at 13.000 rpm for 1 minute.
Hybridization protocol
1. Incubate at 95°C (in heat block), 5 min., spin briefly 2. Place at 42°C (in Maui machine), 5-15 min. 3. Prepare microarray: A)pre-heat array on Maui station B) put array into array-holder (with number facing up going in first) carefully remove glue-sticker from lid and put lid onto array C) take array w/ lid out of array-holder thoroughly attach lid using thick slide place array onto Maui station, approx. 2 min. make sure lid is completely attached D) Add 40 µl hyb solution onto microarray and allow to settle (approx. 15 sec.), dry lid thoroughly 4. Place stickers onto lid, press down on both at the same time, make sure are thoroughly attached 5. Squeeze bubbles away from center of array 6. Wet humid cover and place on Maui station 7. Incubate on Maui at 42°C overnight (17 hours) 8. Remove microarray w/lid from Maui and put in array-holder in Wash 1 (at 42°C) 9. Peel lid off chip while submerged 10. Incubate 10-15 sec. w/ gentle agitation 11. Transfer slide into a slide rack in a new container of Wash 1 (r.t.) Incubate 2 min. w/ gentle agitation 12. Transfer to Wash 2 13. Incubate 1 min. w/ gentle agitation 14. Transfer to Wash 3 15. Incubate 15 sec. w/ gentle agitation 16. Spin approx. 1 min. in Array-Go-Round (place arrays such that numbers are facing to the right when rack is placed in front part of wheel) (the rod on slide holder should be on the inside) 17. Blow off any residual liquid 18. Keep slides protected from light and scan as soon as possible
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.
