|
| Status |
Public on Apr 25, 2010 |
| Title |
total RNA_45' post LPS_experiment 1 |
| Sample type |
RNA |
| |
|
| Source name |
bone marrow derived macrophages from C3H/HeN mice
|
| Organism |
Mus musculus |
| Characteristics |
strain: C3H/HeN mice
cell type: bone marrow cells
|
| Treatment protocol |
Metabolic labelling and purification of nascent RNA were performed essentially as described (Dolken et al., 2008), with minor modifications for use with primary macrophages. In brief, 4 x 107 bone marrow derived macrophages differentiated under the same conditions as for the phosphoproteome analysis (except SILAC) were stimulated on 15 cm dishes with 100 ng/ml LPS Escherichia coli (Sigma Aldrich) for 45 min or 4.5 h or were left untreated. For metabolic labelling, the medium was supplemented with 200 µM 4-thiouridine (4sU, Sigma Aldrich, Cat. No. T4509) during the last 35 min of stimulation.
|
| Growth protocol |
Bone marrow cells from C3H/HeN mice were isolated and cultured in medium for 17 days: After overnight depletion of adherent cells non-adherent cells were expanded by addition of recombinant murine IL-3 (10 mg/L), IL-6 (10 mg/L) and SCF (50 mg/L) (Tebu-Bio) in the presence of 10 % L-cell conditioned medium (LCCM) as a source of macrophage colony stimulating factor (M-CSF) on 10 cm bacteriological plates, starting with 1 x 107 cells per plate. M-CSF was included in the cultures from the beginning to favor the differentiation of macrophages. Cultures were split every 2 to 3 days. After 13 days cells were plated in medium with 10 % LCCM without cytokines to complete differentiation into macrophages for 3 days. On day 16 non-adherent cells were discarded and 25 x 106 adherent cells were plated on 15 cm cell culture plates (Falcon) without LCCM for stimulation the next day.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were lysed by addition of 10 ml Trifast (PeqLab, Germany) and total RNA was extracted. Biotinylation of 4sU labeled RNA was performed in a total volume of 1 ml containing 120 µg RNA, 10 mM Tris (pH 7.4), 1 mM EDTA and 0.2 mg/ml Biotin-HPDP (Pierce) by rotation at RT for 90 min, followed by two rounds of phenol-chloroform extraction and precipitation. For separation of biotinylated nascent and pre-existing unlabelled RNA paramagnetic streptavidin-coated beads and MACS columns were used (Miltenyi, Germany). RNA and beads were mixed and incubated at RT for 15 min, transferred to the columns and washed extensively with washing buffer. The flow through and the first wash volume were collected for recovery of unlabelled, pre-existing RNA. Labelled RNA was eluted with two rounds of 100 µl DTT (100 mM) into buffer RLT (Qiagen, Germany) and cleaned up using RNeasy MinElute Spin Columns. Concentration and integrity of total and labelled RNA were determined by spectrophotometry (Nanodrop) and Experion automated electrophoresis system (Biorad, Munich, Germany).
|
| Label |
biotin
|
| Label protocol |
To investigate the changes in nascent and total mRNA after LPS stimulation of macrophages, RNA samples from two independent experiments were processed and hybridised to Affymetrix Mouse Gene ST 1.0 GeneChips according to the manufacturer’s protocols. In brief, 200 ng total RNA and 100 ng nascent RNA were reverse transcribed introducing by random priming a T7-binding site into the cDNA that allows in vitro transcription. The resulting cRNA was subjected to a second round of random primed cDNA synthesis in the presence of dUTP, that allow fragmentation of the cDNA with uracil DNA glycosylase and apurinic/apyrimidinic endonuclease 1. Biotinylation of the fragmented cDNA was accomplished by incubation with Terminal Deoxynucleotidyltransferase (TdT).
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| |
|
| Hybridization protocol |
5 µg of biotinylated DNA were hybridized to Mouse Gene ST 1.0 GeneChips overnight, followed by washing and staining procedures, and scanning, following Affymetrix protocols.
|
| Scan protocol |
scanning following Affymetrix protocols
|
| Description |
Gene expression data from bone marrow derived macrophages
|
| Data processing |
For generation of probe set expression values, CEL files containing probe level data were normalized using RMA (Affymetrix Expression Console).
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| |
|
| Submission date |
Mar 08, 2010 |
| Last update date |
Jun 03, 2013 |
| Contact name |
Roland Lang |
| E-mail(s) |
[email protected]
|
| Organization name |
University Hospital Erlangen
|
| Street address |
Wasserturmstr. 3-5
|
| City |
Erlangen |
| ZIP/Postal code |
91054 |
| Country |
Germany |
| |
|
| Platform ID |
GPL6246 |
| Series (1) |
| GSE20674 |
Nascent mRNA profiling of LPS-stimulated mouse macrophages |
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