7 protocols
AccessionNameType
P-UMCU-42
feature_extraction
Scanning was performed in a temperature- and humidity-controlled laboratory (20 degrees Celsius, 38% humidity), filtered to remove ozone which was monitored to ensure that this was lower than 5 ppb. Slides were scanned using a G2565BA scanner that has a 48 slide carousel (Agilent, California, USA) at 100% laser power and 30% PMT. After scanning, the intensities for the Cy5 (Red) and Cy3 (Green) channels were automatically extracted using the batch-processing module in ImaGene version 8.0.1 (Biodiscovery, California, USA). For spot finding a local flexibility of 2.0 pixels was used. For segmentation the following settings were used: background buffer: 3.0; background width: 3.0; signal percentages: 3% (low), 97% (high); background percentages: 3% (low), 97% (high). Measurements exported: mean, median, total, standard deviation and area of the foreground and background signals. Batch editor configuration files are available upon request.
P-UMCU-38
labeling
All robotic RNA amplification and labelling procedures were performed in 96 well plates (4titude, Bioke) on a customized Sciclone ALH 3000 Workstation (Caliper LifeSciences) that included a PCR PTC-200 (Bio-Rad Laboratories), SpectraMax 190 spectrophotometer (Molecular Devices), and a magnetic bead-locator (Beckman). For a movie of this automated setup see: http://microarrays.holstegelab.nl/modx/index.php?id=66
RNA amplification
For RNA amplification, total RNA samples were diluted to 0.2 ug/ul and 5 ul is put in a 96-wells plate (Abgene).
All subsequent steps were performed by the following robot script:
1. Mix1 (5 ul) containing 100 ng T7 Mlu VN primer (5’ GGA GGC CGG AGA ATT GTA ATA CGA CTC ACT ATA GGG AGA CGC GTG TTT TTT TTT TTT TTT TTT TTT TTT VN 3’, whereby V= G, A or C; N= G, A, C or T) and external control RNAs, is added to the 5 ul total RNA and mixed in each well. Plate is incubated at 70 degrees C for 10 min and cooled to 48 degrees C.
2. Mix2 containing 4 ul 5x 1st strand buffer, 2 ul 0.1 M DTT (Invitrogen), 1 ul RNAse Inhibitor (Boehringer), 1 ul 20 mM dNTPs (GE Healthcare), 1 ul linear acrylamide, and 1 ul SuperScriptIII (Invitrogen) per sample is prewarmed to 48 degrees C. 10 ul per sample is added and mixed in each well.
3. Plate is incubated at 48 degrees C for 2 hours and cooled to room temperature.
4. 106 ul water and subsequently mix3 containing 15 ul second strand buffer, 3 ul 20 mM dNTPs (GE Healthcare), 1 ul T4 DNA ligase, 4 ul E.coli DNA polymerase I and 1 ul RNAseH (Promega) is added and mixed in each well.
5. Plate is incubated at 16 degrees C for 2 hours, at 65 degrees C for 10 minutes. Double-stranded cDNA product is purified and concentrated with RNAClean (Agencourt, GC biotech) according to manufacturers’ protocol, to an end volume of 25 ul.
6. 8 ul cDNA is put in a 96 well plate and mixed with 12 ul IVT mix containing 2 ul 10x rxn-buffer, 2 ul of each ATP, CTP, GTP, 0.6 ul UTP, 2 ul T7 enzyme mix (MegaScript kit, Ambion, Applied Biosystems), and 2.1 ul 50 mM 5-(3-aminoallyl)-UTP (Ambion, Applied Biosystems).
7. Plate is incubated at 37 degrees C for 4 hours. cRNA product is purified with RNAClean (Agencourt, Beckman) according to manufacturers’ protocol.
8. Concentration is measured (SpectraMax 190) and adjusted to 600 ng/ul. An aliquot of the resulting cRNA plates are sampled QC by Qiaxcel, the remainder is snapfrozen and stored at -80 degrees C.
Labelling
1. NHS-ester Cy3 or Cy5 dye (Amersham PA 23001 and 25001): entire tube is resuspended in 100 ul DMSO (Merck 8.02912.10).
2. 8 ul of each cRNA sample (0.6 ug/ul) is put in a 96-well plate (Abgene), the accompanying common reference sample is put in the next column (final step combines Cy3 and Cy5 labelled material).
3. 3 ul 0.5 M Sodium Bicarbonate buffer, pH 9 is added and mixed to all wells.
4. 3 ul Cy-dye solution is added and mixed to the appropriate wells, plate is incubated at 18 degrees C for 1 hour.
5. 4.5 ul 5 M hydroxylamine is added and mixed, incubated at 18 degrees C for 15 minutes. Labelled cRNA product is purified with RNA Clean (Agencourt, GC biotech) according to manufacturers' protocol.
6. RNA concentration and labelling incorporation are measured (SpectraMax 190).
7. 2.5 ug of each labelled sample and reference cRNA are pooled and subjected to fragmentation according to protocol (Ambion, Applied Biosystems), 15 minutes at 70 degrees C. Samples are stored at -20 degrees C until hybridisation.
P-UMCU-39
hybridization
2.5 ug of each labelled sample in a total volume of 60 ul is combined with 60 ul 2x-hybmix, containing 50% formamide, 10xSSC, 0.2% SDS, 200 ug/ml herring sperm DNA. The hybridisations were performed for 16 hours at 42 degrees Celsius in a HS4800Pro hybstation (Tecan, Männedorf, Switzerland) as detailed below. Hybridisation was performed in a temperature- and humidity-controlled laboratory (20 degrees Celsius, 38% humidity), filtered to remove ozone which was monitored to ensure that this was lower than 5 ppb.
1. 60 ul labelled sample is combined with 60 ul 2x-hybmix, containing 50% formamide, 10xSSC, 0.2% SDS, 200 ug/ml herring sperm DNA.
2. Hybridisations of are performed on an HS4800Pro Hybstation (Tecan, Männedorf, Switzerland).
3. Priming (30 seconds wash and 30 seconds soak) with 5xSSC, 0.1%SDS at 42 degrees Celsius.
4. Injection of pre-hyb mix: 5xSSC, 25% formamide, 0.1%SDS, 1%BSA, total volume 110 ul.
5. Pre-hybridisation: 45 minutes at 42 degrees Celsius, agitation frequency medium, other settings off.
6. Wash in milliQ (2 min wash, 1 min soak), at 42 degrees Celsius, 2x.
7. Wash in 5xSSC, 0.1%SDS (45 secs), at 42 degrees Celsius.
8. Injection of sample. Volume 110 ul.
9. Hybridisation: 16 hours at 42 degrees Celsius, agitation frequency medium, other settings off.
10. Wash (1 min wash, 1 min soak) in 1xSSC, 0.2%SDS at 23 degrees Celsius, 2x.
11. Wash (1 min wash, 1 min soak) in 0.1xSSC, 0.2%SDS at 23 degrees Celsius, 2x.
12. Wash (1 min wash, 1 min soak) in 0.1xSSC at 23 degrees Celsius, 4x.
13. Drying: blow with nitrogen for 3 min at 30 degrees Celsius.
P-MTAB-50
nucleic_acid_extraction
1. Add 1 ml Trizol per 5x 106 cells in suspension (1 ml trizol per 10 cm culture disk) and mix by pipetting, transfer to 1.5 ml eppendorf tube. 2. Incubate 5 min at room temperature. 3. Add chloroform (200 ul per ml Trizol), mix for 15 seconds by shaking vigorously. Incubate 2-3 min at room temperature. 4. Centrifuge 20 min at 14000 rpm 4 degrees C. After centrifugation, transfer the water-phase to a new 1.5-ml micro centrifuge tube. 5. add isopropanol to precipitate RNA. Use 0.5 ml per 1 ml trizol. Mix by shaking, leave samples at RT for 10 minutes. Centrifuge at 14000 rpm for 15 min. 6. wash pellet with 75% ethanol. Centrifuge at 14000 rpm for 15 min. Carefully pipet off all ethanol (respin briefly and use a P20 for the last microliters). Airdry pellets briefly before dissolving in MilliQ water. Depending on pellet-size, this may take a while. Continue with RNeasy columns (Qiagen) for clean-up of RNA (maximal 100 ug/column) 7. Adjust each sample to a volume of 300 ul with sterile MilliQ water. Add 160 ul buffer RLT to the samples, and mix (= lysis buffer). 8. Add 240 ul ethanol (100% stored at -20 degrees C) to the lysate, mix well by pipetting and immediately apply the sample to an RNeasy mini spin column sitting in a 2 ml collection tube (use a second P1000 pipet and work as quick as possible). Next lysate, and so on. 9. Centrifuge at 8000 rpm for 15 seconds in an Eppendorf centrifuge. Discard flowthrough, reuse collection tube. All centrifugation steps at roomtemperature! DNAse treatment: D1. Pipet 350 ul Buffer RW1 into the RNeasy mini column, and centrifuge for 15 s at 8000 rpm to wash. Discard the flow-through. Reuse the collection tube in step D3. D2. Prepare DNase I incubation mix (80 ul/reaction): add 10 ul DNase I stock solution to 70 ul Buffer RDD. Mix by gently inverting the tube. Buffer RDD is supplied with the RNase-Free DNase Set. Note: DNase I is especially sensitive to physical denaturation. Mixing should only be carried out by gently inverting the tube. Do not vortex. D3. Pipet the DNase I incubation mix (80 ul) directly onto the RNeasy silica-gel membrane, and place on the benchtop (20-30 degrees C) for 15 min. Note: Make sure to pipet the DNase I incubation mix directly onto the RNeasy silica-gel membrane. DNase digestion will be incomplete if part of the mix sticks to the walls or the O-ring of the RNeasy column. D4. Pipet 350 ul Buffer RW1 into the RNeasy mini column, and centrifuge for 15 s at 8000 rpm. Discard the flow-through and collection tube. 10. Transfer the RNeasy column into a new 2 ml collection tube. Add 500 ul buffer RPE, wait 1 minute, and centrifuge at 8000 rpm for 15 seconds. Discard flow-through and reuse the collection tube in next steps. 11. Pipet 500 ul buffer RPE onto the RNeasy column, wait 1 minute, and centrifuge at 8000 rpm for 15 seconds. Discard flow-through and reuse collection tube. 12. Centrifuge at 10,000 rpm for 2 minutes (to dry the RNeasy membrane). 13. Transfer the RNeasy column into a new 1.5 ml collection tube. Pipet 100 ul milliQ directly onto the RNeasy membrane and incubate for 4 minutes at room temperature. Centrifuge at 14,000 rpm for 1 minute to elute. Note: When small yields are expected, the elution volume can be lowered to a minimum of 35 ul milliQ. 14. Pipet the eluate of step 13 again onto the RNeasy membrane and incubate for another 4 minutes at room temperature. Centrifuge at 14,000 rpm for 1 minute to elute. After elution, put all RNA-samples on ice. 15. Determine RNA concentration by measuring A260, and RNA intactness by Bioanalyzer. Snap-freeze and store eluate at -80 degrees C.
P-UMCU-40
image_acquisition
Scanning was performed in a temperature- and humidity-controlled laboratory (20 degrees Celsius, 38% humidity), filtered to remove ozone which was monitored to ensure that this was lower than 5 ppb. Slides were scanned using a G2565BA scanner that has a 48 slide carousel (Agilent, California, USA) at 100% laser power and 30% PMT. After scanning, the intensities for the Cy5 (Red) and Cy3 (Green) channels were automatically extracted using the batch-processing module in ImaGene version 8.0.1 (Biodiscovery, California, USA). For spot finding a local flexibility of 2.0 pixels was used. For segmentation the following settings were used: background buffer: 3.0; background width: 3.0; signal percentages: 3% (low), 97% (high); background percentages: 3% (low), 97% (high). Measurements exported: mean, median, total, standard deviation and area of the foreground and background signals. Batch editor configuration files are available upon request.
P-TABM-5694
bioassay_data_transformation
P-TABM-6269
grow
iPS cells were obtained by transduction of OG2 MEFs with a mixture of pMX retroviruses expressing Oct4, Sox2, Klf4, c-Myc, TAF3, TAF4, TAF4b, TAF5, TAF6, TAF7, TAF9, TAF10, TAF11, TAF12, TAF13, and TBP. After 12 days, GFP+ colonies were picked and expanded. Three lines, iPS#1, iPS#4, and iPS#5 were used for microarray analysis. OG2 ES cells or iPS cells derived from OG2 mouse embryonic fibroblasts (MEFs) 3 were cultured on irradiated MEF feeder cells in the following medium: DMEM high glucose (4.5g/l) (Invitrogen), 10% FBS gold (PAA), heat inactivated for 30 min at 56 C, 5% Serum replacement (Gibco), 2 mM L-glutamine (Invitrogen), 0.1 mM non-essential amino acids (Invitrogen), 1 mM sodium pyruvate (Invitrogen), 0.1 mM ?-mercaptoethanol (Sigma), 50 ?g/ml penicillin/streptomycin (Invitrogen), and LIF.