|
| Status |
Public on Mar 14, 2011 |
| Title |
BJ-iPSC replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
BJ-iPSC_2
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: bj-ipsc_2: BJ normal human fibroblasts (CRL-2522) were from ATCC and reprogrammed to iPSC with the protocol defined below
|
| Biomaterial provider |
ATCC
|
| Treatment protocol |
iPSCs Generation. For the generation of human iPSCs, human fibroblasts were seeded in a 6-well plate and spin-infected with a mix of high-quality retroviruses encoding OCT4, SOX2, KLF4, c-MYC, and GFP in the presence of 4 g/ml polybrene. Three infections on consecutive days were performed. 6 days after the first spin-infection, fibroblasts were gently individualized with TrypLE (invitrogen) and seeded onto fresh MEFs in the fibroblast culture medium. After 24 h, the medium was switched to hESC medium. The media was changed every 1–2 days depending on cell density. To establish the iPSC lines, colonies were manually picked and transferred onto MEF feeder cells for several passages before further transfer to Matrigel/mTesR conditions.
|
| Growth protocol |
H9 hESCs (WiCell Research) and iPSCs were maintained on a layer of mitotically inactivated MEFs in DMEM/F12 (Invitrogen) supplemented with 0.1 mM non-essential amino acids (Invitrogen), 1 mM glutamax (Invitrogen), 20% Knockout Serum Replacement (Invitrogen), 55 μM b-mercaptoethanol (Invitrogen) and 10 ng/ml bFGF (Joint Protein Central). hESCs and iPSCs were also cultured in Matrigel (BD Biosciences) with mTeSR medium (Stem Cell Technologies).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using Trizol (Invitrogen) followed by cDNA synthesis using High capability RNA-to-cDNA Mater Mix (Invitrogen).
|
| Label |
Biotin
|
| Label protocol |
The GeneChip microarray processing was performed by the Functional Genomica Core in the Institute for Research in Biomedicine (Barcelona, Spain) according to the manufacturer’s protocols (Affymetrix, Santa Clara, CA). The amplification and labeling were processed as indicated in Nugen protocol with 25ng starting RNA. For each sample, 3.75ug ssDNA were labeled and hybridized to the Affymetrix HG-U133 Plus 2.0 chips.
|
| |
|
| Hybridization protocol |
The GeneChip microarray processing was performed by the Functional Genomica Core in the Institute for Research in Biomedicine (Barcelona, Spain) according to the manufacturer’s protocols (Affymetrix, Santa Clara, CA). The amplification and labeling were processed as indicated in Nugen protocol with 25ng starting RNA. For each sample, 3.75ug ssDNA were labeled and hybridized to the Affymetrix HG-U133 Plus 2.0 chips.
|
| Scan protocol |
Expression signals were scanned on an Affymetrix GeneChip Scanner (7G upgrade). The data extraction was done by the Affymetrix GCOS software v.1.4.
|
| Data processing |
The statistical analysis of the data was performed using ArrayStar 3. Briefly, raw CEL files were imported together with gene annotation from NetAffix (from 11/13/2009) and normalized using RMA algorithm and quantile normalization. As both H9 ESC and HGPS-iPSC originate from female samples, and in order to check for any possible bias introduced by the X and Y chromosome-coded genes, we performed the same analysis with only autosome genes.
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| |
|
| Submission date |
Oct 01, 2010 |
| Last update date |
Mar 14, 2011 |
| Contact name |
Stephanie Boue |
| Organization name |
CMRB
|
| Street address |
Dr Aiguader 88
|
| City |
Barcelona |
| ZIP/Postal code |
08003 |
| Country |
Spain |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE24487 |
Recapitulation of human premature aging by using iPSCs from Hutchinson-Gilford progeria syndrome |
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