|
Status |
Public on Dec 20, 2012 |
Title |
Day6_SSEA1+ M2 [miRNA] |
Sample type |
RNA |
|
|
Channel 1 |
Source name |
Sorted cells
|
Organism |
Mus musculus |
Characteristics |
strain: C57BL6/129 time after induction [days]: 6 cell stage: Intermediates thy1: NEG ssea1: POS 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 8 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 |
|