|
| Status |
Public on Oct 31, 2012 |
| Title |
ESC UNDIFF LINE ES06 PASSAGE 58 |
| Sample type |
RNA |
| |
|
| Source name |
ESC UNDIFF LINE ES06 PASSAGE 58
|
| Organism |
Homo sapiens |
| Characteristics |
class: ESC
condition: UNDIFF
line: ES06
passage: 58
gender: FEMALE
processing control: NA
|
| Treatment protocol |
NA
|
| Growth protocol |
All culture reagents were acquired from Invitrogen unless stated otherwise. Standard culture conditions of 37oC, 5% CO2 and 95% humidity were maintained for all cells. Human ES cells (hESCs) were cultured on a feeder-layer of irradiated CF1 mouse embryonic fibroblasts (MEFs) in DMEM:F12 (Cat# 11330-032) containing 20% Knockout Serum Replacement (KSR)(Cat# 10828-028), 1mM glutamine (Cat# 25030-081), 0.1mM beta-mercaptoethanol (beta-ME; Sigma), 1x non-essential amino acids (NEAA; Cat# 11140-050) and 4ng/ml bFGF (R&D Systems)(Cat# 233-FB). Fibroblasts were cultured in DMEM (Cat# 11965-092) containing 10% fetal bovine serum (FBS) (Gemini Bio-products), 2mM glutamine and 1x NEAA. Fibroblasts were irradiated with ~6500 rads using a Faxitron RX650 X-irradiator. They were subsequently plated on Falcon 6-well tissue culture dishes, coated with 0.1% gelatin, at a density of 0.1875 x 106/well. hESCs were plated in small clumps the following day, medium was exchanged every day and colonies were passaged by collagenase treatment every 3-4 days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the mirVana RNA Isolation kit (Ambion) as recommended by the vendor for array-based assays. Concentration was determined using a NanoDrop ND-1000 UV-VIS Spectrophotometer.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled miRNA was prepared from 0.1 ug RNA using the miRNA Labeling Reagent and Hybridization kit (Agilent) according to the manufacturer's instructions, followed by purification using Micro Bio-spin 6 columns (Bio-Rad).
|
| |
|
| Hybridization protocol |
The entire purified labeled miRNA sample was dried using a speed-vac on medium setting and resuspended in 18ul of DNase/RNase-free water. To each sample, 4.5ul of 10x GE Blocking Agent and 22.5ul of 2x Hybridization Buffer (Agilent) were added. Samples were gently vortexed, incubated at 100oC for 5 minutes and chilled in an ice water bath for 5 minutes. The entire volume of each sample was hybridized to Agilent Human miRNA microarray kit (G4470A) for 20 hours at 55°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 5 minutes at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent).
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 8x15k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, 5uM scanning mode ‘Single Pass’, eXtended Dynamic range selected, Dye channel set to Green and Green PMT set to XDR high 100%, XDR low 10%).
|
| Description |
ESC UNDIFF LINE ES06 PASSAGE 58
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol miRNA-v1_95_May07 and Grid: 016436_D_20070426) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Dec 13, 2011 |
| Last update date |
Nov 01, 2012 |
| Contact name |
Kory R Johnson |
| E-mail(s) |
johnsonko@ninds.nih.gov
|
| Phone |
301-402-1956
|
| Organization name |
NINDS/NIH
|
| Department |
DIR IT & Bioinformatics
|
| Lab |
Bioinformatics Section
|
| Street address |
10/3B01, 9000 Rockville Pike
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL9081 |
| Series (2) |
| GSE34199 |
The NIH Human Pluripotent Stem Cell Database (Agilent, miRNA) |
| GSE34200 |
The NIH Human Pluripotent Stem Cell Database |
|
