Am2238

CLIP was performed as described (41 (link)) with the following modifications. Briefly, two 15 cm dishes of HEK293T cells per CLIP were transfected with pEGFPC1-hLa (42 (link)), or indicated GFP-hLa variants or myc-tagged pcDNA-PABPC1 (43 (link)) (PolyJet, SignaGen). Twenty four hours post-transfection, cells were UV-crosslinked (Stratalinker at 254 nm, 1000 mJ) and cells were lysed in RIPA buffer (10 mM Tris–HCl pH8.0, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl, 1 mM PMSF). Lysates were then treated with 3 μl of 1:100 RNAse I (100 U/μl AM2294 Invitrogen) or 40 μl of a 1:100 dilution of RNase T1 (1 U/μl AM2283 Ambion) and 2 μl DNase I (2 U/μl AM2238 Ambion) at 37°C for 3 min. Antibodies used were anti-cmyc (Abcam ab21060) or anti-GFP (Abcam ab1218). coIPs were performed using Protein G magnetic Dynabeads (Invitrogen). Antibodies were incubated with Dynabeads in 150 μl RIPA buffer for 1 h at room temperature, then antibodies/Dynabeads were incubated with lysates for 2 h at 4°C. Complexes were washed 2× with RIPA buffer and 3× with proteinase K buffer prior to digestion with 10 μl proteinase K (buffer and enzyme supplied by Invitrogen, #100005393 20 mg/ml). Eluted RNA was probed with ³²P radiolabeled dT (40 (link)), stripped and reprobed for pre-tRNA Met-e (probe sequence AAA TTA TTG TGC CCC G) via northern blot.

After RNA isolation using RNeasy minikit (Qiagen, 74104) followed by 30 min of DNase treatment (Ambion, AM2238) at 37°C, poly(A)⁺ RNA transcript was isolated [NEBNext poly(A) mRNA magnetic isolation module; New England Biolabs, E7490] from 1 µg of total RNA for RNA library preparation and sequencing using NEBNext Ultra directional RNA library preparation kit for Illumina (New England Biolabs, E7420S) according to the manufacturer's instruction. The samples were sequenced on an Illumina HiSeq 2500 with 100-bp paired-end reads at the Vincent J. Coates Genomics Sequencing Laboratory at the University of California at Berkeley.