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| Status |
Public on Aug 05, 2010 |
| Title |
S2-DRSC (Celniker/RNA:483) extraction2_array1 |
| Sample type |
RNA |
| |
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| Source name |
S2-DRSC (Celniker/RNA:483) extraction2_array1 channel_1
|
| Organism |
Drosophila melanogaster |
| Characteristics |
cell line: S2-DRSC
tissue: embryo-derived cell-line
developmental stage: late embryonic stage
sex: Male
|
| Growth protocol |
Description of the growth and maintenance of S2-DRSC cells used by modENCODE for RNA preparations.
|
| Extracted molecule |
nuclear RNA |
| Extraction protocol |
For isolation of nuclear RNA from cell cultures. Nuclei are isolated by differential centrifugation and RNA is collected using the TRIzol protocol for cell lines. This protocol is modified from Zimmerman et al 1982 (PNAS 79:3148) and used to prepare nuclear RNA from cell lines
cDNA Synthesis and Labeling: Note that this protocol gives you labeled dsDNA, as such the array results are not stranded.* ug cDNA hybed per chip: 10 ug if template was total RNA or Poly-A-, 2.5 ug if Poly-A+ RNAFIRST-STRAND SYNTHESIS - 10 ug of Total RNA (DNAsed,Cleaned-up and ETOH precipitated) - 1.25 ug of 250ng/ul random primers (Invitrogen) - diH2O to 60 ul Vf.Incubate at 70C for 10 mins (fast ramp) then cool to 15C over the course of 20 minutes. Once it reaches 15C add: - 20 ul of 5X First-Strand Buffer (Invitrogen) - 10 ul of 100mM DTT (Invitrogen) - 5 ul of 10mM dNTPs (Invitrogen)Incubate at 15C for 30 minutes then heat to 42C over the course of 20 minutes. Once it reaches 42C add: - 10 ul Superscript RT II (Invitrogen)Incubate at 42C for 60 minutes, then heat rapidly to 70C and incubate for 15 minutes, then park it at 4C.SECOND-STRAND SYNTHESISTo the First strand synthesis reaction above add while at 4C: - 450 ul diH2O - 150 ul 5X Second-Strand Buffer (Invitrogen) - 15 ul 10mM dNTPs (Invitrogen) - 5 ul 10U/ul E. coli DNA Ligase (Invitrogen) - 20 ul 10U/ul E. coli DNA Polymerase (Invitrogen) - 5 ul 2U/ul E. coli RNAseH (Invitrogen)Incubate at 16C for 120 minutes, then 70C for 15 minutes, then park it at 4C.RNA REMOVAL and cDNA PURIFICATIONAdd: - 3 ul of 10U/ul RNaseH (Ambion) - 3 ul of 5 and 20U/ul RNAseA/T1 (Ambion)Incubate at 37C for 20 minutesUse a Qiagen PCR Clean-up kit (two columns per reaction), ETOH precipitate the eluate and 70% ETOH rinse. Redissolve in 35 ul diH2O. Measure the OD600 on a Nanodrop.cDNA FRAGMENTATION*** NEED TO ADD ***Check the fragmentation on an agarose gel. Want it to be around 100 nucleotides.
|
| 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. TdT LABELING; Perform in 20 ul for 5 ug of DNA, 35 ul for 10 ug of DNA, and 70 ul for 10-15 ug of DNA. - 40 ul of fragmented DNA - 14 ul of 5X TdT buffer (Roche) - 7 ul of 20mM CoCl2 (Roche) - 2.3 ul of DNA Labeling Reagents (Affymetrix) - 0.5 ul of Terminal deoxytransferase, 8000U (Roche) - 6.2 ul of diH2OIncubate at 37C for 2 hours in water bath.The labeled cDNA is now ready to be hybridized.
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|
| 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.
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| Scan protocol |
Scanned at 0.7 microns/pixel.
|
| Data processing |
Data_Normalization_for_Expression_Arrays protocol. 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 Signal Graph generation protocol. 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
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| Submission date |
Jul 28, 2010 |
| Last update date |
Aug 05, 2010 |
| Contact name |
DCC modENCODE |
| E-mail(s) |
help@modencode.org
|
| 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
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| City |
Toronto |
| State/province |
Ontario |
| ZIP/Postal code |
M5G 0A3 |
| Country |
Canada |
| |
|
| Platform ID |
GPL6629 |
| Series (1) |
| GSE23226 |
Dm_S2-DRSC_nuclearRNA_p200_482-483-484_38bp |
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