|
| Status |
Public on Aug 01, 2011 |
| Title |
HP1 gamma -/-_E11.5_PGCs_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
HP1g-/-, E11.5, PGCs, replicate 1
|
| Organism |
Mus musculus |
| Characteristics |
cell type: primordial germ cells (PGCs)
Stage: embryonic day 11.5 (E11.5)
genotype: HP1 gamma -/-
|
| Treatment protocol |
The genital ridges of HP1g-/-; Blimp1-mVenus tg mice and wild-type; Blimp1-mVenus tg mice were dissected from embryos and singly cells were suspended in FACS solution (2% FCS and 0.01% NaN3 in Hank’s solution). The mVenus-positive cells were isolated by a flow cytometer (JSAN, Bay Bioscience)
|
| Growth protocol |
HP1g mutant mice were generated by a modified gene-trap method. To mark the PGCs by fluorescence, HP1g+/- mice were mated with Blimp1-mVenus transgenic mice and HP1g+/-; Blimp1-mVenus tg mice were backcrossed to HP1g+/- mice to make HP1g-/-; Blimp1-mVenus tg mice that were used.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The total RNA was extracted from these sorted cells by an RNeasy micro kit (Qiagen). RNA was quantified and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 4 ng of total RNA using the One-Color Low Input Quick Amp Labeling kit (Agilent) according to the manufacturer's instructions with some changes. 4th Spike mix was made by diluted 3rd Spike-Mix 20 times, and 1.6 ul of 4th Spike-Mix was added to samples. Cy3 labeled cRNA was synthesized at 40°C for 20 hours and purified by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
0.9 ug of Cy3-labelled cRNA (specific activity >7.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Mouse Whole Genome Microarray 4x44K (G4122F) for 17 hours at 65°C 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), then dried by raise it slowly from buffer 2.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green. Scan mode is eXtended Dynamic range Scan mode and PMT is set to 10% and 100%).
|
| Description |
Gene expression of HP1g-/- PGCs at E11.5
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid: 014868_D_F_20100617) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Feb 14, 2011 |
| Last update date |
Aug 01, 2011 |
| Contact name |
Masahide Asano |
| E-mail(s) |
asano@kiea.m.kanazawa-u.ac.jp
|
| Phone |
+81 76-265-2463
|
| Fax |
+81 76-234-4240
|
| Organization name |
Kanazawa University
|
| Department |
Advanced Science Research Center,
|
| Lab |
Division of Transgenic Animal Science
|
| Street address |
13-1
|
| City |
Kanazawa |
| State/province |
Ishikawa |
| ZIP/Postal code |
920-8640 |
| Country |
Japan |
| |
|
| Platform ID |
GPL4134 |
| Series (1) |
| GSE27294 |
The effect of HP1-gamma deficiency on primordial germ cells |
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