|
| Status |
Public on May 23, 2018 |
| Title |
IgG control-RIP RNA from Dicer KO BMDMs |
| Sample type |
RNA |
| |
|
| Source name |
IgG control-immunoprecipitated RNA from Dicer KO macrophages
|
| Organism |
Mus musculus |
| Characteristics |
genotype: LysM-Cre/Dicerflox/flox/Apoe–/–
tissue: bone marrow
cell type: macrophage
age: 6-8 weeks
Sex: Male
antibody: IgG control
|
| Growth protocol |
mouse bone marrow-derived macrophages were cultured in DMEM-F12/10% FCS/10% L929-conditioned medium, at 37°C in a humidified atmosphere of 5% CO2
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Macrophages were washed with ice-cold PBS, subsequently incubated for 15 min on ice in lysis buffer (100 mM KCl, 5 mM MgCl2, 10 mM HEPES pH 7.0, 0.5% NP40, 5 mM DTT, 250 units/ml RNase OUT, 400 μM vanadyl ribonucleoside complexes, and 1 tablet per 10 ml protease inhibitors), and homogenized with a Dounce homogenizer. The lysates were centrifuged at 16,000 x g for 15 min at 4°C. The input RNA (without immunoprecipitation) was extracted from the supernatant with TRIzol. Prior to the immunoprecipitation, protein A/G-conjugated magnetic beads were incubated with a mouse monoclonal anti-AGO2 antibody or IgG control. The antibody-conjugated beads were subsequently incubated with the cell extracts in the supernatant for 7 hours at 4°C before immobilization of the precipitate with a magnetic separator. RNA was isolated from the precipitate with TRIzol, reverse-transcribed and pre-amplified with Ovation PicoSL WTA System V2.
|
| Label |
Cy3
|
| Label protocol |
500 ng cDNA of each sample were subjected to restriction digestion with a combination of Alu I and Rsa I. The digested cDNA samples were directly labeled with exo-Klenow fragment and random primers by incorporation of Cyanine-3 dUTP.
|
| |
|
| Hybridization protocol |
Following cRNA clean-up and quantification, 530 to 600 ng of each Cyanine-3-labeled sample was fragmented and prepared for one-color-based hybridization (Gene Expression Hybridization Kit, Agilent Technologies). Labeled samples were hybridized at 65°C for 17 hrs on separate Agilent SurePrint G3 Mouse Gene Expression 8x60K Microarrays (AMADID 028005). Afterwards, microarrays were washed with increasing stringency using Gene Expression Wash Buffers (Agilent Technologies) followed by drying with acetonitrile (SIGMA).
|
| Scan protocol |
Fluorescence signal intensities were detected with Scan Control A.8.4.1 software (Agilent Technologies) on the Agilent DNA Microarray Scanner.
|
| Description |
IgG-immunoprecipitated RNA
|
| Data processing |
The scanned images were analyzed with Feature Extraction 10.7.3.1 software (Agilent Technologies) and the design files 028005_D_F_20131202.xml to obtain background subtracted and spatially detrended Processed Signal intensities.
|
| |
|
| Submission date |
Oct 06, 2016 |
| Last update date |
May 23, 2018 |
| Contact name |
Andreas Schober |
| E-mail(s) |
aschober@med.lmu.de
|
| Organization name |
Ludwig-Maximilians-University
|
| Department |
Institute for Cardiovascular Prevention
|
| Street address |
Pettenkoferstr. 9
|
| City |
Munich |
| State/province |
Bavaria |
| ZIP/Postal code |
80336 |
| Country |
Germany |
| |
|
| Platform ID |
GPL13912 |
| Series (2) |
| GSE87717 |
Ago2-RIP-chip in Dicer WT and KO macrophages |
| GSE87721 |
Ago2-RIP-chip and miRNA expression profile in Dicer WT and KO macrophages from bone marrow or aorta |
|
