|
| Status |
Public on Mar 30, 2012 |
| Title |
HGPS iPSC replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
HGPS-iPSC_2
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: iPSC
lmna mutation status: before correction
|
| Biomaterial provider |
Coriell Cell Repositories http://ccr.coriell.org/Sections/Search/Search.aspx?PgId=165&q=AG01972 http://ccr.coriell.org/Sections/Search/Search.aspx?PgId=165&q=AG04110
|
| 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 ug/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.
Targeted gene correction of LMNA mutations in HGPS-iPSC and AWS-iPSC were accomplished using Helper-Dependent Adenovirus (HDAd)-based gene correction vector.
|
| Growth protocol |
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). iPSCs were also cultured in Matrigel (BD Biosciences) with mTeSR medium (Stem Cell Technologies).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using TRIzol reagent according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA) and purified further using the Rneasy Mini Kit (Invitrogen).
|
| Label |
Biotin
|
| Label protocol |
Affymetrix GeneChip® IVT Labeling Kit, according to manufactuer's protocol (GeneChip® Expression Analysis Technical Manual)
|
| |
|
| Hybridization protocol |
Affymetrix Eukaryotic Target Hybridization protocol (GeneChip® Expression Analysis Technical Manual)
|
| Scan protocol |
Affymetrix® GeneChip® Scanner 3000 with GCOS software according to manufactuer's protocol (GeneChip® Expression Analysis Technical Manual)
|
| Description |
cell culture
|
| Data processing |
Probe set summarization and normalization was performed by robust multi-array averaging (RMA, including background subtraction, summarization by median polish and quantile normalization) using the R/Bioconductor package affy.
|
| |
|
| Submission date |
Apr 13, 2011 |
| Last update date |
Mar 30, 2012 |
| Contact name |
Sachin Kumar |
| E-mail(s) |
skumar@salk.edu
|
| Organization name |
Salk Institute for Biological Studies
|
| Department |
Gene Expression Laboratory
|
| Lab |
GEL-B
|
| Street address |
10010 N.Torrey Pines Road
|
| City |
San Diego |
| State/province |
California |
| ZIP/Postal code |
92037 |
| Country |
USA |
| |
|
| Platform ID |
GPL571 |
| Series (1) |
| GSE28607 |
Targeted gene correction of LMNA mutations in patient-specific iPSCs |
|
