Primary mouse embryonic fibroblasts were isolated as described previously (Todaro G.J. and Green H. (1963), J. Cell Biol., 17, 299-313; Brusselbach S.U. et al. (1995), Oncogene, 10, 79-86.)
Growth protocol
Cells were grown using high glucose-DMEM (Invitrogen) with 1% Penicillin/Streptomycin (Invitrogen) and 10% dialyzed FBS (cutoff 10,000Da, Sigma) to near confluency. Cells were serum-starved for 48h with 1% dialyzed FBS, and then incubated for 2h with media containing 10% FBS (stimulation control).
Extracted molecule
total RNA
Extraction protocol
100 ug/ml medium of cycloheximide were then added and cells further incubated at 37C for 30min. Cells were washed with PBS containing 100ug/ml cycloheximide, trypsinized (Trypsin-EDTA, 100ug/ml cycloheximide), washed again with PBS and centrifuged at 1000rpm for 6min at 4C. Cells were then lysed by NP-40 lysis buffer (1ml for ~ 107 cells; 10mM Tris-HCl pH 8, 150mM NaCl, 1.5mM MgCl2, 0.1% (v/v) NP-40, 100ug/ml cycloheximide, 0.5mg/ml heparin, 1mM DTT) and douncing. The lysate was centrifuged with 10,000xg at 4C for 10min to remove nuclei and mitochondria. The supernatant was added onto a linear sucrose gradient (15%-50% sucrose) performed in a Beckman SW40 tube using a Biocamp Gradient Master (Nycomed Pharma) based on 10mM Tris-HCl pH 7.5, 140mM NaCl, 1.5mM MgCl2, 1mM DTT and either 15% or 50% sucrose. Gradients were then centrifuged using an SW 40 rotor at 4C with 40,000xg for 2h. The polysomal profile was analyzed at 260nm and fractionated with an ISCO density fractionator (ISCO Inc.) coupled to a UV cell. mRNAs associated with either no, or one ribosomal subunit, or only 1 full ribosome were referred to as 'undertranslated'. Fractions were collected in Eppendorf tubes containing SDS (final concentration: 1% SDS) and precipitated using PelletPaint (Novagen). Equal fractions of different biological and technical replicates were pooled and RNA eluted using the RNeasy MINI cleanup procedure (QIAGEN) to get rid of the remaining heparin. Samples were then phenol-chloroform (PC, pH 6.6) extracted using PLG heavy 0.5ml Eppendorf tubes, followed by a final precipitation with PelletPaint, 3M NaAcetate and 100% Ethanol and a wash step with 70% Ethanol. The RNA pellet of each fraction was diluted in 10ul water. RNA yield and quality were measured by the RNA6000 Nano assay (Bioanalyzer 2100, Agilent Technologies).
Label
biotin
Label protocol
RNA was reverse-transcribed, linearly amplified as described previously (Kenzelmann M. et al. (2004), Genomics 83, 550-558.), and biotin-labelled via a T7-based strategy according to manufacturer's instructions (Affymetrix).
Hybridization protocol
Following fragmentation, 10microg of cRNA were hybridized to MU74Av2 GeneChip (Affymetrix) according to manufacturer's instructions.
Scan protocol
GeneChips were scanned using the GeneChip Scanner 3000 7G System (Affymetrix).
Description
Gene expression data from primary serum treated MEF cells grown to near confluency.
Data processing
The data were analyzed with the SAS MicroArray Solution 1.3, using standard settings except the following: consistence within experimental groups (stimulus and labelling combination) was determined by array group correlation. Loglinear mixed models with Bonferroni corrections were fitted for values of perfect-matches, only with stimulus (10% FBS) and labelling (s4U) considered to be constant and the chip-ID as random.
