R1 Mouse Embryonic Stem Cell poly(A)+ RNA at 24 hrs
Extracted molecule
polyA RNA
Extraction protocol
To generate poly(A)+ RNA, R1 Mouse Embryonic Stem (R1mES) cells were cultured over mouse Embryonic Fibroblast (MEF) cells mitotically arrested with gamma-radiation. Cells were maintained in serum-free media supplemented with 15% ES-grade serum (Invitrogen), L-glutamine, Penicillin-streptomycin, beta-marcaptoethanol and leukemia inhibitory factor (Chemicon). All cells were routinely harvested at ~ 70% confluency and Poly(A)+ RNA was enriched from isolated total RNA using the Oligotex Kit (Qiagen) following manufacturer’s recommendation. Three independent samples of Poly(A)+ RNA was prepared from separate cultures of R1mES cells. First-strand cDNA synthesis was performed by using SuperScript II reverse transcriptase in the reaction volume of 105 ul for 15 ug of starting RNA material. The RNA was mixed with random hexamers (83.3 ng/g mRNA), heated to 700C for 10 min, and cooled to 150C after which 5x SuperScript II First Strand buffer, DTT (10 mM), and dNTPs (0.5 mM) were added. SuperScript II was added after a 20-min incubation (200 units/ug RNA) followed by a 20-min ramp to 420C and 60-min incubation at 420C. SuperScript II was inactivated at 750C for 15 min. The second-strand cDNA was synthesized by addition of 50 units of Escherichia coli DNA ligase, 200 units of E. coli DNA polymerase I, 10 units of E. coli RNase H, and 0.2 mM dNTPs to the first-strand synthesis reaction at 160C for 2 h. Double-stranded cDNA was treated with RNase H (Epicentre Technologies) and RNases A/T1 (Ambion), extracted by using a QIAquick PCR purification kit (Qiagen), and subjected to further fragmentation to 50-100 bp by DNase I (1 unit/ul; Epicentre Technologies; size distribution of fragmented DNA was verified on a 2% agarose gel).
Label
Affymetrix DNA labeling reagent (#900542).
Label protocol
The fragmented cDNA was then end-labeled with 2.5mM biotinylated DNA labeling reagent (Affymetrix) by using 100 units of terminal deoxynucleotidyltransferase (TdT; Roche Diagnostics) in 1x TdT buffer (Roche Diagnostics) and 5 mM CoCl2 (Roche Diagnostics) for 2 h at 370C.
Hybridization protocol
Finally 2ug of the labeled DNA material was hybridized per chip to Affymetrix Mouse Tiling Array 1.0R Array (16 chip set) for 18 h at 450C in a 3 M tetramethyl ammonium chloride/1x MES-based solution containing 100microg/mL Herring Sperm DNA, 0.02% Triton and 30pM of biotinylated control oligo B2 (Affymetrix). All reagents were from Invitrogen, except where noted otherwise.
Scan protocol
N/A
Description
C004
Data processing
Processing of the microarray data to generate graphs representing a plot of intensities of each probe mapping to the genome versus its genomic coordinate was performed in the following steps : (1) All the three independent biological replicas from all four time points (00,12,24,and 168hrs) were quantile normalized together and then scaled to have a median feature intensity of 25 (2) The Perfect match (PM), mismatch (MM) intensities for each probe was then mapped to the genome using exact 25mer matching. For each genomic position to which a probe pair matched a data set was generated containing all PM, MM probes within a window of + 50 base pairs. Using a pseudo-median approach the PM-MM data were calculated in a sliding window across the genome as an estimator of the expression level at each genomic position. In order to extract usable positive probes from this data a pseudo-median threshold corresponding to a false positive rate of 2.9% for each time point was estimated from the bacterial probes tiled on the chip. Probes that are above this threshold were considered as present and the rest were noted as absent. Columns #2 and #3 are obtained from analysis done on mm5.
