cell line: AINV15 cell type: murine ESC line with controllable Esrrb expression (Esrrb_R) dox withdrawal: day0
Treatment protocol
To induce differentiation, we withdrew Doxycycline (Dox) (1 µg ml-1, Sigma) from the media while maintaining all other routine ESC nutrients and harvested cells at Day1, Day3 and Day5
Growth protocol
A murine ESC line with controllable Esrrb expression (Esrrb_R) was maintained on irradiated primary mouse embryonic fibroblasts (MEFs). For all experiments, ESCs were cultured on 0.1% gelatin-coated tissue culture plates without feeder cells. Esrrb_R cell line was maintained in media containing Glucose (Dulbecco’s modified Eagle’s medium-1X-High Glucose) (Gibco®, Invitrogen), 15% FBS (Fetal bovine serum) (Hyclone, Thermo Scientific), 100 mM MEM non-essential amino acids, 0.1 mM 2-mercaptoethanol, 1 mM L-glutamine, Penicillin/Streptomycin (Gibco, Invitrogen) and 103 U ml-1 LIF (Chemicon, Millipore) and Doxycycline (Dox) (1 µg ml-1, Sigma) D-MEM-High . All cell cultures were maintained at 37C with 5% CO2 and cells were plated at a density of 3×10^5 cells per 10 cm dish.
Extracted molecule
total RNA
Extraction protocol
Total RNA extracted using Trizol following manufacturer's instructions
Label
Hy3
Label protocol
1000 ng total RNA from sample and reference was labelled with Hy3™ and Hy5™ fluorescent label, respectively, using the miRCURY™ LNA Array power labelling kit (Exiqon, Denmark) following the procedure described by the manufacturer.
sample type: reference (pool of Esrrb Day0 + Day 1 samples)
Treatment protocol
To induce differentiation, we withdrew Doxycycline (Dox) (1 µg ml-1, Sigma) from the media while maintaining all other routine ESC nutrients and harvested cells at Day1, Day3 and Day5
Growth protocol
A murine ESC line with controllable Esrrb expression (Esrrb_R) was maintained on irradiated primary mouse embryonic fibroblasts (MEFs). For all experiments, ESCs were cultured on 0.1% gelatin-coated tissue culture plates without feeder cells. Esrrb_R cell line was maintained in media containing Glucose (Dulbecco’s modified Eagle’s medium-1X-High Glucose) (Gibco®, Invitrogen), 15% FBS (Fetal bovine serum) (Hyclone, Thermo Scientific), 100 mM MEM non-essential amino acids, 0.1 mM 2-mercaptoethanol, 1 mM L-glutamine, Penicillin/Streptomycin (Gibco, Invitrogen) and 103 U ml-1 LIF (Chemicon, Millipore) and Doxycycline (Dox) (1 µg ml-1, Sigma) D-MEM-High . All cell cultures were maintained at 37C with 5% CO2 and cells were plated at a density of 3×10^5 cells per 10 cm dish.
Extracted molecule
total RNA
Extraction protocol
Total RNA extracted using Trizol following manufacturer's instructions
Label
Hy5
Label protocol
1000 ng total RNA from sample and reference was labelled with Hy3™ and Hy5™ fluorescent label, respectively, using the miRCURY™ LNA Array power labelling kit (Exiqon, Denmark) following the procedure described by the manufacturer.
Hybridization protocol
The Hy3™-labeled samples and a Hy5™-labeled reference RNA sample were mixed pair-wise and hybridized to the miRCURY™ LNA Array version 5th Generation (Exiqon, Denmark), which contains capture probes targeting all miRNAs for human, mouse or rat registered in the miRBASE version 14.0 at the Sanger Institute. The hybridization was performed according to the miRCURY™ LNA array manual using a Tecan HS4800 hybridization station (Tecan, Austria).
Scan protocol
The microarray slides were scanned and stored in an ozone free environment (ozone level below 2.0 ppb) in order to prevent potential bleaching of the fluorescent dyes. The miRCURY™ LNA array microarray slides were scanned using the Agilent G2565BA Microarray Scanner System (Agilent Technologies, Inc., USA)
Description
1_Exiqon*.txt and 0_Exiqon*.txt contain raw data from ch1 (Hy3) and ch2 (Hy5) sample, respectively.
Data processing
The image analysis was carried out using the ImaGene 8.0 software (BioDiscovery, Inc., USA). The quantified signals were background corrected (Normexp with offset value 10 - Ritchie et al., 2007) and normalized using the global Lowess (LOcally WEighted Scatterplot Smoothing) regression algorithm. Values that should be disregarded (e.g., failed, not detected') are labeled as "null".
