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Status |
Public on Apr 27, 2015 |
Title |
kh2_36hrs_rep3 |
Sample type |
RNA |
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Source name |
KH2 cells, 36 hrs RA
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Organism |
Mus musculus |
Characteristics |
cell type: kh2 timepoint: 36hrs after RA
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Treatment protocol |
ES Cells were differentiated with differentiation media (DMEM + 10% Serum + NAA+ 0.03µM RA) RA supplemented 33nm for requisite length of time. Feeders were separated by plating freshly trypsinized cells on gelatinized plate for 30 min. After half an hour media was aspirated and centrifuged for 5 min at 1000 rpm.
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Growth protocol |
KH2 cells at Passage 12 were cultured on gamma irradiated feeder cells with DMEM containg 15% fetal bovine serum, NAA and β-mercaptoethanol.
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Extracted molecule |
total RNA |
Extraction protocol |
RNA isolation was performed using Trizol and later purified by RNA Easy Kit (Quiagen). Total RNA in the amount of 1ug was amplified according to Ambion’s Message Amp II aRNA Amplification Kit, part number AM1751. Positive control RNA from Agilent’s One Color RNA Spike-In Kit, part number 5188-5282, was used to monitor sample amplification and labeling as well as array hybridization.
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Label |
Cy3
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Label protocol |
Amplified mRNA, referred to as aRNA, was quantified on a NanoDrop ND-1000 and a mass of 2ug was labeled with cy3 dye using Kreatech’s ULS Fluorescent Labeling Kit, part number EA-023.
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Hybridization protocol |
Labeled aRNA was quantified on the NanoDrop ND-1000 and a 1.5µg mass of cy3 labeled aRNA was hybridized to custom Agilent 2x105K HOX tiling arrays. Hybridizations were performed at 65 degrees C for 17 hours under standard conditions (1X Agilent blocking agent, and 1X Agilent hybridization buffer) and slides were washed successively with Agilent GE wash buffer 1, at room temperature and with Agilent GE wash buffer 2, at 31oC, prior to scanning.
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Scan protocol |
Microarray images were acquired with an Agilent High-Resolution DNA Microarray Scanner (G2505C). Hybridization, array washing, scanning and probe information extraction with Agilent’s Feature Extraction Software (Version 10.5.1.1) were all performed according to Agilent’s One-Color Microarray-Based Gene Expression Analysis Protocol, Version 6.0, December 2009 (Low Input Quick Amp Labeling).
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Data processing |
Agilent tiling arrays were hybridized in a single-color configuration and data was read into R. Agilent gMeanSignal was used as the measurement (mean green signal for each spot). Data was analyzed using the limma package. Data was normalized between arrays using scale normalization. Replicates were averaged and bedgraph files were created and visualized using IGV and UCSC genome browser.
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Submission date |
Sep 29, 2014 |
Last update date |
Apr 27, 2015 |
Contact name |
Madelaine Gogol |
Organization name |
Stowers Institute
|
Department |
Computational Biology Core
|
Street address |
1000 E. 50th Street
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City |
Kansas City |
State/province |
MO |
ZIP/Postal code |
64110 |
Country |
USA |
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Platform ID |
GPL19214 |
Series (1) |
GSE61585 |
Retinoids induce rapid dynamic changes in the non-coding RNAs and epigenetic profiles of murine Hox clusters (tiling) |
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