|
| Status |
Public on Oct 31, 2012 |
| Title |
iPSC UNDIFF LINE i12 PASSAGE 10 |
| Sample type |
RNA |
| |
|
| Source name |
iPSC UNDIFF LINE i12 PASSAGE 10 (SAMPLE 88)
|
| Organism |
Homo sapiens |
| Characteristics |
class: iPSC
condition: UNDIFF
line: i12
passage: 10
gender: FEMALE
processing control: SAMPLE 147
|
| 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. Embryoid bodies were cultured in fibroblast medium (FBS; EB_mesend) or in hESC medium without bFGF (EB_ecto) in 60mm Corning Low Attachment dishes for a total of 8 days. Media were changed by sedimentation every 2 days. An important point to note is that the same lot number of FBS was used for all studies.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using a modification of the basic Trizol (Invitrogen) protocol. Briefly, 1ml of Trizol was added to sedimented colonies or EBs and triturated to dissociate the cells. At this point the lysates were stored at -80oC until all samples for that cell line were collected. Upon thaw, lysates were incubated at room temperature for 10 mins, mixed with 200ul chloroform and centrifuged in a Phase-Lock Gel (Heavy) Eppendorf tube (Qiagen). RNA was precipitated from the aqueous phase by the addition of 250ul of isopropanol and 250ul of a high salt buffer (0.8 M sodium citrate and 1.2 M NaCl) followed by centrifugation. The RNA pellet was washed twice with 75% ethanol, dried and resuspended in nuclease-free water. RNA was DNase treated for 20 mins and the DNAse removed using Ambion’s DNA-Free kit. Concentration was determined using a NanoDrop ND-1000 UV-VIS Spectrophotometer.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.2 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.65 ug of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 55ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturer’s instructions. On completion of the fragmentation reaction, 55ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112F) for 17 hours at 65°C 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 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 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to XDR high 100%, XDR low 10%).
|
| Description |
iPSC UNDIFF LINE i12 PASSAGE 10
Raw data file: SAMPLE 88.txt
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v5_91 and Grid: 014868_D_20070820) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Oct 12, 2011 |
| Last update date |
Oct 31, 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 |
GPL6480 |
| Series (2) |
| GSE32923 |
The NIH Human Pluripotent Stem Cell Database (Agilent, mRNA) |
| GSE34200 |
The NIH Human Pluripotent Stem Cell Database |
|
