GSM3178972: Snap25_1_CAATTCG_TRAP_RF; Mus musculus; OTHER
TRAP-RF-in-vivo: P21 Snap25::eGFP-RpL10a, Aldh1l1:: eGFP-RpL10a, along with their eGFP-negative littermates were euthanized, and their brains frozen in liquid nitrogen and stored at -80C until use. Two brains were pooled per sample, and replicate experiments were done. TRAP was  performed as described (Heiman et al 2008) with a few modifications. Briefly, the brains were homogenized in ice in a buffer (20 mM pH 7.4 HEPES, 150 mM KCl, 5 mM MgCl2, 0.5 mM dithiothreitol, 100 μg/ml CHX, Turbo DNAse, protease inhibitors, and RNase inhibitors). The lysates were cleared by centrifuging at 2000 xg for 10 m at 40C and then treated with DHPC (to 30mM, Avanti) and NP-40 (to 1%, Ipgal-ca630, Sigma) for 5 min in ice. Lysates were then further cleared by centrifuging at 20,000 xg for 15 m at 40C and then mixed with protein L-coated magnetic beads (Invitrogen), previously conjugated with a mix of two monoclonal anti-GFP antibodies  (Doyle et al 2008), and incubated with rotation for 4 h at 4°C. Beads were washed 5 times with a high-salt buffer (20 mM pH7.4 HEPES, 350 mM KCl, 5 mM MgCl2, 1% NP-40, 0.5 mM dithiothreitol, and 100 μg/ml CHX) and then resuspended in normal-salt buffer (150 mM KCl, otherwise as above). To couple to RF, on-bead RNA digestion was performed with RNAse I (Invitrogen) for 1 h with end-to-end rotation, followed by washing three times with normal-salt buffer. Small ribosomal subunits with the mRNA fragments were eluted with ribosome dissociation buffer as described (20 mM pH7.3 Tris-HCl, 250 mM NaCl, 0.5% Triton X-100, 50mM EDTA) (Gonzalez et al 2014). RNA was extracted with phenol-chloroform, quality-tested with Agilent BioAnalyzer, and then subjected to dephosphorylation and subsequent library preparation as described for RF (Ingolia et al 2011). Briefly, the cell lysates were treated with RNAseI and subjected to sucrose-cushion ultracentrifugation for pelleting monosomes. mRNA fragments were isolated from the monosomes, size-selected in a polyacrylamide, ligated to cloning linkers, and converted to cDNAs. The cDNAs were then circularized, depleted of rRNAs, PCRed and size-selected and finally deep-sequenced on Illumina Hiseq 2000 (50 bp, single end).