|
| Status |
Public on Jul 25, 2016 |
| Title |
MEF_KO_Contact_inhibition_dyeswap |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Embryonic fibroblast cells derived from LAT2-/- C57BL/6J mice
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J
genotype: Lats2-/-
condition: low density
|
| Treatment protocol |
Cells were cultured for 48h until subconfluet and for 3 - 5 days prior to RNA sampling for low density sample and high density sample respectively.
|
| Growth protocol |
Mouse embyonic fibroblasts (MEFs) were cultured in DMEM + 10% inactivated fetal bovine serum (FBS), penicillin (100U/mL), and streptomycin (100μg/mL) at 37°C, 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from cultured cells with miRNeasy Mini Kit (Qiagen) and processed according to the manufacturer’s protocol. The quality of the RNAs was estimated by using the RNA 6000 Nano LabChip Kit (p/n 5065-4476) on the Agilent 2100 Bioanalyzer (G2940BA; Agilent Technologies, Santa Clara, CA).
|
| Label |
cy5
|
| Label protocol |
500 ng of total RNA was labeled using Quick-Amp Labeling Kit (Agilent Technologies, Pato Alto, CA, USA). In brief, cDNA was reverse transcribed from 500ng of total RNA with an oligo-dT-T7 promoter primer and MMLV-RT. Second strand synthesis was carried out with random hexamers. Fluorescent antisense cRNA was synthesized with cyanine 5-CTP (Cy5-CTP) or 3-CTP (Cy3-CTP), and T7 polymerase. The fluorescent-labeled antisense cRNA was purified with miRNeasy columns (Qiagen) according to manufacturer's protocols and resuspended in water. The purified products were quantified at A650nm for Cy5-CTP and at A552nm for Cy3-CTP with Agilent 2100 Bioanalyzer (Agilent Technologies).
|
| |
|
| Channel 2 |
| Source name |
Embryonic fibroblast cells derived from LAT2-/- C57BL/6J mice
|
| Organism |
Mus musculus |
| Characteristics |
cell type: embryonic fibroblasts
strain: C57BL/6J
genotype: Lats2-/-
cell type: embryonic fibroblasts
condition: high density
|
| Treatment protocol |
Cells were cultured for 48h until subconfluet and for 3 - 5 days prior to RNA sampling for low density sample and high density sample respectively.
|
| Growth protocol |
Mouse embyonic fibroblasts (MEFs) were cultured in DMEM + 10% inactivated fetal bovine serum (FBS), penicillin (100U/mL), and streptomycin (100μg/mL) at 37°C, 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from cultured cells with miRNeasy Mini Kit (Qiagen) and processed according to the manufacturer’s protocol. The quality of the RNAs was estimated by using the RNA 6000 Nano LabChip Kit (p/n 5065-4476) on the Agilent 2100 Bioanalyzer (G2940BA; Agilent Technologies, Santa Clara, CA).
|
| Label |
cy3
|
| Label protocol |
500 ng of total RNA was labeled using Quick-Amp Labeling Kit (Agilent Technologies, Pato Alto, CA, USA). In brief, cDNA was reverse transcribed from 500ng of total RNA with an oligo-dT-T7 promoter primer and MMLV-RT. Second strand synthesis was carried out with random hexamers. Fluorescent antisense cRNA was synthesized with cyanine 5-CTP (Cy5-CTP) or 3-CTP (Cy3-CTP), and T7 polymerase. The fluorescent-labeled antisense cRNA was purified with miRNeasy columns (Qiagen) according to manufacturer's protocols and resuspended in water. The purified products were quantified at A650nm for Cy5-CTP and at A552nm for Cy3-CTP with Agilent 2100 Bioanalyzer (Agilent Technologies).
|
| |
|
| |
| Hybridization protocol |
Before hybridization, 825ng labeled cRNA of each product was fragmented and mixed with control targets and hybridization buffer according to the manufacturer's protocol (Agilent Technologies). Hybridizations were done for approximately 17h at 65°C. The slides were washed according to the manufacturer's manual, and scanning of microarrays was performed with 5µm resolution using a DNA microarray laser scanner (Agilent Technologies).
|
| Scan protocol |
The array was scanned using Agilent G2505C DNA microarray scanner. Images were quantified using Agilent Feature Extraction Software (version 10.5.1.1).
|
| Description |
KO_Contact_inhibition_dyeswap
|
| Data processing |
Agilent Feature Extraction Software (v 10.5.1.1) was used for background subtraction, LOWESS normalization, and dye-normalization .
|
| |
|
| Submission date |
Nov 21, 2014 |
| Last update date |
Jul 25, 2016 |
| Contact name |
Daisuke Okuzaki |
| E-mail(s) |
dokuzaki@biken.osaka-u.ac.jp
|
| Phone |
+81-6-6879-4935
|
| Organization name |
Osaka univ.
|
| Department |
Immunology Frontier Research Center
|
| Lab |
Human Immunology （Single Cell Genomics)
|
| Street address |
Yamadaoka 3-1
|
| City |
Suita |
| State/province |
Osaka |
| ZIP/Postal code |
565-0871 |
| Country |
Japan |
| |
|
| Platform ID |
GPL4134 |
| Series (2) |
| GSE63535 |
Transcriptome profiling of mouse embryonic fibroblasts upon contact inhibition |
| GSE63538 |
LATS2 regulates repressive epigenetic integrity via regulation of Polycomb repressive complex 2 |
|
