|
| Status |
Public on May 11, 2018 |
| Title |
Ezh2-0h-rep2 (part 2) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Ezh2-CD4cre-KO naïve CD8+ T-cells unstimulated
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Ezh2-c-KO CD8+ T-cell
time point: 0h
|
| Treatment protocol |
Ezh2 (Ezh2fl/fl) mice were obtained from MMRRC repository, CD4Cre mice was obtained from Taconic, and OT-I mice from Jackson Laboratory. Ezh2fl/fl mice were crossed with CD4Cre to generate the Ezh2fl/fl-CD4Cre+(Ezh2-c-KO) strain. Ezh2fl/fl-CD4Cre mice were further crossed with OT-I to generate Ezh2fl/fl-CD4Cre -OT-I (Ezh2-c-KO OT-I) strain. All mice were maintained under specific pathogen free conditions at the animal facility of National Institute on Aging and animal care was conducted in accordance with the guidelines of NIH.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were cultured in RPMI-1640 with 10% FBS, 10mM HEPES, 0.11nM beta-metcaptoethanol and 1x Pen/Strep/Glu from Thermo-Fisher. Naive CD8+ T cell stimulations performed using plate coated αCD3 (2C11) and soluble αCD28 (37.51) (Biolegend). Total RNA was extracted with RNaeasy Kit on QIAcube (Qiagen).
|
| Label |
Cy3
|
| Label protocol |
RNA was labeled using Quick Amp Labeling two-color kit (Agilent) and hybridized to SurePrint G3 Mouse Gene Expression v2 8x60K Microarray Kit (Agilent) according to manufacturer’s protocols.
|
| |
|
| Channel 2 |
| Source name |
Mix of resting and activated mouse T cells
|
| Organism |
Mus musculus |
| Characteristics |
biomaterial provider: Home-made
extract protocol: Total RNA was extracted with RNaeasy Kit on QIAcube (Qiagen)
|
| Treatment protocol |
Ezh2 (Ezh2fl/fl) mice were obtained from MMRRC repository, CD4Cre mice was obtained from Taconic, and OT-I mice from Jackson Laboratory. Ezh2fl/fl mice were crossed with CD4Cre to generate the Ezh2fl/fl-CD4Cre+(Ezh2-c-KO) strain. Ezh2fl/fl-CD4Cre mice were further crossed with OT-I to generate Ezh2fl/fl-CD4Cre -OT-I (Ezh2-c-KO OT-I) strain. All mice were maintained under specific pathogen free conditions at the animal facility of National Institute on Aging and animal care was conducted in accordance with the guidelines of NIH.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were cultured in RPMI-1640 with 10% FBS, 10mM HEPES, 0.11nM beta-metcaptoethanol and 1x Pen/Strep/Glu from Thermo-Fisher. Naive CD8+ T cell stimulations performed using plate coated αCD3 (2C11) and soluble αCD28 (37.51) (Biolegend). Total RNA was extracted with RNaeasy Kit on QIAcube (Qiagen).
|
| Label |
Cy5
|
| Label protocol |
RNA was labeled using Quick Amp Labeling two-color kit (Agilent) and hybridized to SurePrint G3 Mouse Gene Expression v2 8x60K Microarray Kit (Agilent) according to manufacturer’s protocols.
|
| |
|
| |
| Hybridization protocol |
Slides were hybridized to SurePrint G3 Mouse Gene Expression v2 8x60K Microarray (Agilent) according to manufacturer’s protocols
|
| Scan protocol |
The hybridized chips were scanned with SureScan Microarray Scanner D (Agilent) and the expression level was determined with Agilent Feature Extraction software 11.5.1.1.
|
| Description |
Ezh2-CD4cre-KO naïve CD8+ T-cells unstimulated, replication 2
|
| Data processing |
Data are extracted with Agilent Feature Extraction Software. The data were further processed with ExAtlas. See https://lgsun.irp.nia.nih.gov/exatlas for details.
The data is normalized with the following method:
(1) Take values from the columns gProcessedSignal, rProcessedSignal in the raw files and log-transform them using: log10(max(x,10)). These values will be referenced below as Xgi and Xri, where i is array number.
(2) Take average of Xri's for the oligo among 12 arrays in the series: AverXr = average(Xri).
(3) For each array, estimate Yi = Xgi-Xri+AverXr (adjust to UMR).
|
| |
|
| Submission date |
Nov 02, 2017 |
| Last update date |
May 11, 2018 |
| Contact name |
Minoru S.H. Ko |
| E-mail(s) |
[email protected]
|
| Phone |
410-558-8359
|
| Organization name |
NIH
|
| Department |
National Institute on Aging
|
| Lab |
Lab of Genetics
|
| Street address |
251 Bayview Blvd, Suite 100, 10C
|
| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21224 |
| Country |
USA |
| |
|
| Platform ID |
GPL10787 |
| Series (2) |
| GSE106425 |
Ezh2 is essential for activation-induced CD8+ T cell cycle progression via repressing Cdkn1a and Cdkn2c expression (Part II) |
| GSE106426 |
Ezh2 is essential for activation-induced CD8+ T cell cycle progression via repressing Cdkn1a and Cdkn2c expression |
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