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Status |
Public on Nov 30, 2010 |
Title |
M4+ |
Sample type |
RNA |
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Source name |
Tis21–GFP positive S phase cells, replicate 3
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Organism |
Mus musculus |
Characteristics |
strain: C57BL/6J developmental stage: Embryonic day (E) 14.5 tissue: dorsal telencephalon (cerebral cortex) cell cycle: S phase genotype/variation: Tis21 positive
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Treatment protocol |
Dorsal telencephalon (cerebral cortex) was dissected from E14.5 Tis21-GFP heterozygous mouse embryos in Tyrode’s solution. The tissue was digested with trypsin mix solution (0.13 % trypsin (Sigma–Aldrich), 0.067 % Hyaluronidase (Sigma–Aldrich) and 0.02 % kynurenic acid (Sigma–Aldrich) in serum-free medium consisting of DMEM and F-12 nutrient (1:1; Invitrogen) for 20 min at 37°C. 0.07 % trypsin inhibitor was added to stop the digestion, collect cells at 600 g for 5 min in a centrifuge, dissociate cells in 0.07 % trypsin inhibitor, washed cells with DMEM and F-12 medium and then filtrate cells to obtain single cells. Single cells were incubated with a vital dye (Vybrant DyeCycle Violet stain, Invitrogen) at 37°C for 1 hour and sorted S phase cells by DNA contents between 2N and 4N DNA mass using a FACSAria II cell sorter (Becton, Dickinson).
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Growth protocol |
All mouse embryos used were heterozygous Tis21-GFP knock-in mice (C57BL/6 background), obtained by overnight mating of homozygous Tis21-GFP males with wildtype C57BL/6 females. Noon of the day on which the vaginal plug was observed was defined as embryonic day (E) 0.5. All animal studies were performed in strict pathogen-free conditions in the animal facility of the Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.
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Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was prepared using NucleoSpin RNA XS kit (Macherey-Nagel) following the manufacturer's recommendations. RNA was quantified and quality controlled using the RNA 6000 Pico Kit on the Agilent 2100 Bioanalyzer RNA (Agilent Technologies).
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Label |
Cy3
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Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 50 ng total RNA using the Low Input Quick Amp Labeling kit (Agilent) according to the manufacturer's instructions in the Agilent One-Color Microarray-Based Gene Expression Analysis v.6.5 Manual. Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
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Hybridization protocol |
0.6 ug of Cy3-labelled cRNA (specific activity >12.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 ul of 2x GEx Agilent hybridization buffer HI-RPM was added to the fragmentation mixture and hybridized to Agilent SurePrint G3 Mouse GE 8x60K Microarrays (G4858A-028005) for 17 hours at 65°C and 10 rpm in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent). The wash buffers contained 0.005% Triton X-102.
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Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using the AgilentG3_GX_1Color scan protocol for 8x60K high densitiy Micorarray Formats (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%, the resulting file is a 20bit tiff image).
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Description |
Gene expression after FACS in the S phase cells
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Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.1.1 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid: 028005_D_F_20100617) to obtain background subtracted and spatially detrended green Processed Signal intensities. Normalized signal intensity (log2 transformed and quantile normalized) calculated from the green processed signal after Feature Extraction using the GeneSpring software version 11.0 (Agilent)
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Submission date |
Aug 12, 2010 |
Last update date |
Nov 30, 2010 |
Contact name |
Yoko Arai |
E-mail(s) |
arai@mpi-cbg.de
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Organization name |
Max Planck Institute
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Department |
MPI-CBG
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Lab |
Huttner lab
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Street address |
Pfotenhauerstrasse 108
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City |
Dresden |
ZIP/Postal code |
01307 |
Country |
Germany |
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Platform ID |
GPL10787 |
Series (1) |
GSE23585 |
Neural stem and progenitor cells shorten S-phase upon commitment to neuron production |
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