|
| Status |
Public on Dec 20, 2012 |
| Title |
Day3_Thy+ M3 [mRNA] |
| Sample type |
RNA |
| |
|
| Source name |
Sorted cells
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL6/129
time after induction [days]: 3
cell stage: Intermediates
thy1: POS
ssea1: NEG
gfp: NEG
|
| Treatment protocol |
Harvested cells were incubated with antibodies against Thy1.2 (PE conjugated, 53-2.1, eBiosciences) and SSEA-1 (mouse IgM, MC-480, Developmental Hybridoma Bank) for 20 minutes. Cells were washed in PBS and then incubated for 20 minutes with APC conjugated anti mouse IgM, (eBioscience) and Pacific Blue-conjugated streptavidin (Invitrogen). The cells were washed in PBS, resuspended in propidium iodide 5% FBS/PBS solution and passed through a 40mm cell strainer to achieve single cell suspension. Cells were sorted on a FACSAria (BD Biosciences ) and/or analysed in a LSRII (BD Bioscience). For analysis and/or sorting of intermediates, cells were stained with Thy1.2 and SSEA1 antibodies and sorted or analyzed as indicated.
|
| Growth protocol |
Mouse Embryonic fibroblast (MEFF) cultures were established from E13.5 embryos from a reprogrammable mice strain carrying one copy of the OKSM cassette and Rosa26-M2rtTA allele (het/het) or carrying two copies of the OKSM cassette and Rosa26-M2rtTA allele (ho/ho). Reprogramming was performed in ESC medium (KODMEM with 15% FBS, L-Glutamin, penicillin-streptomycin, non-essential amino acids, b-mercaptoethanol and 1000 U/ml LIF) in the presence of doxycycline.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
iPSCs were harvested when they reached about 50% confluency and preplated on non-gelatinized T25 flasks for 45 minutes to remove feeder cells. Intermidiates cell types were harvest after FACS sorting. Cells were then spun down and the pellet used for isolation of total RNA using the miRNeasy Mini Kit (QIAGEN) without DNase digestion. RNA was eluted from the columns using 50 ml RNase-free water or TE buffer, pH7.5 (10 mM Tris-HCl and 0.1 mM EDTA) and quantified using a Nanodrop (Nanodrop Technologies).
|
| Label |
biotin
|
| Label protocol |
Samples were labeled with biotin in the standard way
|
| |
|
| Hybridization protocol |
Samples were hybridized to affymetrix MOE430A arrays using the protocol provided by the supplier
|
| Scan protocol |
Arrays were scanned on an affymetrix scanner in according to the manufacturers protocol
|
| Description |
Sample name: KH182
|
| Data processing |
Data were normalized using robust multi array averaging (RMA) and converted to log2 scale
|
| |
|
| Submission date |
Nov 19, 2012 |
| Last update date |
Dec 20, 2012 |
| Contact name |
Ben S. Wittner |
| E-mail(s) |
[email protected]
|
| Organization name |
Massachusetts General Hospital
|
| Department |
Center for Cancer Research
|
| Lab |
Lawrence
|
| Street address |
149 13th Street
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02129 |
| Country |
USA |
| |
|
| Platform ID |
GPL8759 |
| Series (2) |
| GSE42379 |
Defining a molecular roadmap of cellular reprogramming into iPS cells [mRNA profiling] |
| GSE42478 |
Defining a molecular roadmap of cellular reprogramming into iPS cells |
|
