|
| Status |
Public on Dec 20, 2012 |
| Title |
Day9_Thy- M2 [miRNA] |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Sorted cells
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL6/129
time after induction [days]: 9
cell stage: Intermediates
thy1: NEG
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 |
RNA was isolated using the miRNeasy Mini Kit (QIAGEN)
|
| Label |
Cy5
|
| Label protocol |
Each sample was labeled using the miRCURY™ Hy3™/Hy5™ power labelling kit
|
| |
|
| Channel 2 |
| Source name |
a pool of all the samples
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL6/129
sample type: reference
|
| 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 |
RNA was isolated using the miRNeasy Mini Kit (QIAGEN)
|
| Label |
Cy3
|
| Label protocol |
Each sample was labeled using the miRCURY™ Hy3™/Hy5™ power labelling kit
|
| |
|
| |
| Hybridization protocol |
Each hybridized on Exiqon BWA 0.5.9 arrays against a pool of all the samples.
|
| Scan protocol |
Samples were scanned as a service by Exiqon using their inhouse protocol
|
| Description |
Sample 10
0_Exiqon* files are channel1 raw data files.
1_Exiqon* files are channel2 raw data files.
|
| Data processing |
Samples were loess normalized and probes with more than 3 missing values were removed from further consideration. Other missing data were imputed using a KNN method
|
| |
|
| Submission date |
Nov 23, 2012 |
| Last update date |
Dec 20, 2012 |
| Contact name |
Ben S. Wittner |
| E-mail(s) |
wittner.ben@mgh.harvard.edu
|
| 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 |
GPL14957 |
| Series (2) |
| GSE42475 |
Defining a molecular roadmap of cellular reprogramming into iPS cells [miRNA profiling] |
| GSE42478 |
Defining a molecular roadmap of cellular reprogramming into iPS cells |
|
