|
| Status |
Public on Jun 02, 2009 |
| Title |
ML-DmD8 (Celniker/RNA:208) extraction2_array1 |
| Sample type |
RNA |
| |
|
| Source name |
ML-DmD8 (Celniker/RNA:208) extraction2_array1 channel_1
|
| Organism |
Drosophila melanogaster |
| Characteristics |
cell line: ML-DmD8
tissue: dorsal mesothoracic disc
genotype: y v f mal
|
| Extracted molecule |
total RNA |
| Extraction protocol |
This protocol is used by the IU Bloomington labs to prepare RNA from cell lines that will be used for for microarray hybridization or RT-PCR.This is a standard TRIzol purification procedure, followed by DNase and RNeasy (Qiagen). Concentration was determined by Nanodrop absorbance.
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 70C for 10 min, and cooled to 15C 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 42C and 60-min incubation at 420C. SuperScript II was inactivated at 75C 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 16C for 2 h.
|
| Label |
Biotin
|
| Label protocol |
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).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 37C.
|
| |
|
| Hybridization protocol |
For array hybridization, 2ug of the labeled DNA material was hybridized per chip for 18 h at 45C in a 3 M tetramethyl ammonium chloride/1x MES-based solution containing 100?g/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 |
Scanned at 0.7 microns/pixel.
|
| Description |
n/a
|
| Data processing |
Three biological replicates were hybridized for this experiment. The data from replicate arrays were quantile-normalized (Bolstad et al., 2003) and all arrays were scaled to a median array intensity given (e.g. 361). Processed data are obtained using following parameters: median value is 361 The sliding window approach (bandwidth 50) has been used to estimate RNA abundance (signal which is listed in column #2). It was found by calculating the median of all pairwise average PM-MM values, where PM is a perfect match and MM is a mismatch. Processed data are obtained using following parameters: bandwidth is 50 genome version is r5
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| |
|
| Submission date |
May 28, 2009 |
| Last update date |
Jun 02, 2009 |
| Contact name |
DCC modENCODE |
| E-mail(s) |
[email protected]
|
| Phone |
416-673-8579
|
| Organization name |
Ontario Institute for Cancer Research
|
| Lab |
modENCODE DCC
|
| Street address |
MaRS Centre, South Tower, 101 College Street, Suite 800
|
| City |
Toronto |
| State/province |
Ontario |
| ZIP/Postal code |
M5G 0A3 |
| Country |
Canada |
| |
|
| Platform ID |
GPL6629 |
| Series (1) |
| GSE16283 |
Dm_DmD8_TotalRNA_p200_206-208-210_38bp |
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