The mRNA tagging protocol is a modification of the protocol by Goodwin et al. 1. For each sample, 1 ml of packed worms were resuspended in M9, and then fixed in 0.5% formaldehyde in M9 buffer for 1 hour at 4oC. Animals were rinsed in homogenization buffer (HB), and then resuspended in 3 mls of HB. HB is prepared by DEPC treating 300 mM NaCl, 50 mM Hepes pH 7.6, 10 mM MgCl2, 1 mM EGTA, 30 mM EDTA, 0.2 mg/ml Heparin (sodium salt), and 10% Glycerol, and then adding DTT to 1 mM, vanadyl ribonucleoside complex to 8 mM (Sigma), 50 U RNasin/ml, and ½ protease inhibitor cocktail tablet (Roche). Animals were lysed with 2-3 passes on a French press at 8000 PSI, and then 25 passes on a Wheaten homogenizer. Large debris was sedimented by centrifugation at 4300 x g for 6 minutes, and then smaller debris was separated from the supernatant by centrifugation at 48000 x g for 20 minutes. At this point, the lysate could be flash frozen and stored for future use.
RNA bound to the FLAG::PAB-1 was enriched using anti-FLAG-M2 affinity gel beads (Sigma). The affinity beads were prepared by rinsing two times in RNase-free glycine HCl (pH 3.5) and then four times in HB at 4 °C. The beads were collected by centrifugation, and stored at 4 °C. 1 ml of lysate, 50U of RNasin, 8 ul of 200 mM vanadyl ribonucleoside, and 75 ul of pelleted anti-FLAG-M2 affinity gel beads were mixed together for 2-3 hours at 4oC. The affinity beads were washed four times with cold HB, and then the RNA-protein cross links were reversed by incubating the beads in 125 ul of elution buffer (50 mM Tris/HCl pH8.0, 10 mM EDTA, 1.3% SDS; 20U Rnasin) at 65oC for 30 minutes. The supernatant containing the RNA was collected, the elution was repeated once more and then the two supernatants were combined.
RNA was isolated by mixing 250 ul of the immunoprecipitation supernatant (or cell extract) with 1 ml of trizol, and then mixing 250 uls of chloroform. After letting the mixture stand for 10 minutes, the mixture was spun in a microfuge at 14,000 RPM for 15 minutes at 4oC, and then the supernatant was extracted with chloroform. RNA was precipitated by adding 600 ul of isopropanol, incubating at room temperature for 10 minutes and then centrifuging in a microfuge for 20 minute at 14,000 RPM at 4oC. The RNA pellet was rinsed twice with 1 ml of 70% ETOH.
RNA was linearly amplified as previously described 2. Labeled cDNA was synthesized using 5 to 20 ug of amplified RNA, and primed with 5 ug of random hexamers and 2 ug of anchored oligo-d(T) primers. A description of the DNA microarrays, probe preparation, and microarray hybridization are described in Jiang et al., 2000. The amplified RNA from the cell extract was used to prepare Cy-3 labeled cDNA and the amplified co-immunoprecipitated RNA was used to prepare Cy-5 labeled cDNA.
1. Goodwin, E. B., Okkema, P. G., Evans, T. C. & Kimble, J. Translational regulation of tra-2 by its 3' untranslated region controls sexual identity in C. elegans. Cell 75, 329-39 (1993).
2. Wang, E., Miller, L. D., Ohnmacht, G. A., Liu, E. T. & Marincola, F. M. High-fidelity mRNA amplification for gene profiling. Nat Biotechnol 18, 457-9 (2000).