To distinguish between proteomes of OPs, IOs, and OCs, we performed 3-plex SILAC and used mass spectrometry to quantify the proteome of each cell type. RAW 264.7 cells were subcultured in phenol red–free custom-made Alpha Minimum Essential Medium, as phenol red has an estrogen activity (20) that is reported to suppress RANKL-induced osteoclast formation, and arginine (Arg) and lysine (Lys) were replaced by 12C6-Arg and 14N2 12C6-Lys (light, Sigma), 13C6-Arg and 2H4-Lys (medium, Cambridge Isotope Laboratories, Tewksbury, MA), or 13C6 15N4-Arg and 13C6 15N2-Lys (heavy, Cambridge Isotope Laboratories). After these isotopes were fully incorporated into the cellular proteins (roughly four passages; each sample of medium- and heavy-labeled cells was checked to measure the label incorporation percentage), 412,300 cells of light-, medium-, and heavy-labeled cells were seeded onto 0.1% gelatin coated10-cm dishes (density: 7,500 cells/cm2) on day 0. Both medium- and heavy-labeled cells were treated with 40 ng/ml RANKL (R&D Systems, Minneapolis, MN) on day 1, and the medium was replaced with fresh medium with RANKL on day 3.The cells were harvested on day 5 post-treatment.
Growth protocol
RAW 264.7 cells were routinely maintained in phenol red–free Alpha Minimum Essential Medium (Invitrogen, Carlsbad, CA) containing 10% FBS (HyClone), 100 U/ml penicillin, 100 g/ml streptomycin, and 25 ng/ml amphotericin B (Invitrogen) and cultured on 0.1% gelatin-coated (Sigma) dishes.
Extracted molecule
total RNA
Extraction protocol
RNA was purified by Qiagen RNAeasy kit
Label
biotin
Label protocol
Illumina TotalPrep RNA Amplification Kit (Ambion)
Hybridization protocol
Standard Illumina hybridization protocol
Scan protocol
Standard Illumina scanning protocol with Illumina HiScan-SQ
Description
_6297001019_B
Data processing
Signal data was extracted from the image files with the Gene Expression module (v. 1.9.0) of the GenomeStudio software (v. 2011.1) from Illumina, Inc. Signal intensities were converted to log 2 scale. Probes that did not have detection p-value < 0.1 in at least 2 arrays were removed from the dataset. After filtering probes, quantile normalization was applied across all arrays.
