GSM2303118: FL_RPF 3; Mus musculus; OTHER
For Ribo-Seq, the cell pellet was re-suspended in 200 μl of cold lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 15 mM MgCl2, 100 μg/ml cycloheximide, 1 mM DTT, 1% Triton X-100, 8% glycerol, 20 U/ml TURBO™ DNase (Ambion, AM1907)) and incubated for 30 min at 4°C with occasional vortexing. The lysate was clarified by sequential centrifugation for 5 min at 1800 x g and 10000 x g at 4°C to remove nuclei and mitochondria. The lysate was then treated with RNase I (Ambion, AM2294) for 30 min at RT to digest mRNAs not protected by the ribosome. The digestion was stopped by adding 4.5 μl of SUPERaseIn™ RNase Inhibitor (20 U/μl, Ambion AM2696). Lysate was then loaded onto a 1 M sucrose cushion. Ribosomes were pelleted by ultracentrifugation at 70000 rpm for 4 hr at 4°C by TLA120.2 rotor. The pellet was re-suspended in Trizol (Invitrogen, 15596) and RNAs were extracted by following manufacturer’s protocol. For RNA-Seq, after centrifugation, the cell pellet was re-suspended in Trizol (Invitrogen, 15596) for RNA extraction. RNA was extracted and polyA mRNA was isolated using Oligotex mRNA Mini Kit (Qiagen, 70022) following manufacturer’s protocol. Purified mRNAs were fragmented in alkaline fragmentation buffer (100 mM NaCO3 pH9.2, 2 mM EDTA). Briefly, ribosome protected fragments (RPFs) and fragmented RNAs were loaded onto a 15% urea gel. 28-31 nt RPFs and 30-50 nt fragmented RNAs were excised from the gel for Ribo-Seq and RNA-Seq respectively. RNAs were eluted, dephosphorylated by PNK (NEB, M0201S), and ligated to the miRNA Cloning linker (NEB, S1315S) by T4 RNA Ligase2 truncated K227Q (NEB, M0242S). Ligated RNA was gel purified and reverse transcribed by Superscript III (Invitrogen, 18080). Gel purified cDNAs were circularized by Circligase (Epicentra, CL4111K) and rRNA sequence were subtracted using biotinylated oligos. Amplification was done using Phusion High Fidelity DNA Polymerase (NEB, M0530S). PCR amplification was performed for 10-15 cycles and products were loaded onto non-denaturing 8% PAGE gel. DNA fragment were purified for Illumina sequencing.