|
| Status |
Public on Jul 26, 2018 |
| Title |
Oct4-GFPpos spermatogonia rep 1 |
| Sample type |
RNA |
| |
|
| Source name |
Oct4-GFPpos spermatogonia
|
| Organism |
Mus musculus |
| Characteristics |
strain background: mixed C57BL/6 x CBA
tissue: Testis
cell type: Oct4-GFPpos spermatogonia
|
| Treatment protocol |
Testis cell suspensions were prepared by a standard two step digestion protocol with collagenase and trypsin. Cells were sorted on a BD Influx Cell Sorter (BD Biosciences). The undifferentiated population was gated according to expression of the Plzf-mCherry/CreER reporter plus surface markers c-Kit and CD9. Plzf is a well-established marker of undifferentiated spermatogonia plus cells at early differentiation stages. c-Kit marks differentiating spermatogonia and CD9 is enriched on functional stem cells. Undifferentiated cells were then isolated according to whether they were positive or negative for Oct4-GFP expression.
|
| Growth protocol |
Adult transgenic mice (6-8 weeks old) containing both Oct4-GFP and Plzf-mCherry/CreER reporters were maintained under standard conditions.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Sorted cells were lysed in TRIzol LS reagent (Thermo Fisher Scientific) then RNA was purified and DNase treated using a Direct-zol RNA Miniprep kit (Zymo Research).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labelled cRNA was prepared from 0.1ug total RNA using the One-Color Low input Quick Amp labelling Kit (Agilent, v6.6 September 2012) according to the manufacturer’s instructions, followed by RNeasy column purification (Qiagen). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
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| |
|
| Hybridization protocol |
600ng of Cy3 labeled cRNA (specific activity > 6 pmol Cy3/ug cRNA) was fragmented at 60oC for 30 minutes in a reaction volume of 25ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturer’s instructions. On completion 25ul of 2x Agilent gene expression hybridization buffer was added and 43ul of sample hybridised for 17 hours at 67oC 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 37oC GE wash buffer 2(Agilent).
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent C, DNA microarray scanner using one color scan settings for 8x60k array slides; scan area 61x21.6mm, scan resolution 3um, dye channel is set to Green and 20 bit Tiff.
|
| Description |
#3 070414.1 GFP +
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 11.0.1.1 (Agilent) using default parameters (protocol GE1-1100_Jul11 and Grid: 028005_D_F_20120201) to obtain background subtracted and spatially detrended Processed Signal intensities.
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| |
|
| Submission date |
Feb 08, 2017 |
| Last update date |
Jul 26, 2018 |
| Contact name |
Robin Mark Hobbs |
| E-mail(s) |
robin.hobbs@monash.edu
|
| Organization name |
Hudson Institute of Medical Research
|
| Department |
Centre for Reproductive Health
|
| Lab |
Germline Stem Cell Biology Laboratory
|
| Street address |
27-31 Wright Street
|
| City |
CLAYTON |
| State/province |
VIC |
| ZIP/Postal code |
3168 |
| Country |
Australia |
| |
|
| Platform ID |
GPL13912 |
| Series (2) |
| GSE94647 |
Analysis of gene expression in populations of adult undifferentiated spermatogonia [microarray] |
| GSE107256 |
Analysis of gene expression in populations of adult undifferentiated spermatogonia |
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