GSM3702258: sh-Scram_Ribo_seq_Rep1; Homo sapiens; OTHER
293T sh-Scramble and sh-YTHDC2 Cells were lysed in lysis buffer (10 mM HEPES, pH 7.4, 100 mM KCl, 5 mM MgCl2, 1% Triton X-100) containing CHX (100 µg ml-1),The supernatant was collected and subjected to sucrose gradient sedimentation. For RNA-seq, 12 µg Trizol extracted total RNAs were fragmentated by using freshly prepared RNA fragmentation buffer (10 mM Tris-HCl, pH 7.0, 10 mM ZnCl2) and heating at 94°C for 5 min, followed by adding 1 µl 0.5 M EDTA to  terminate. For Ribo-seq, *E. coli* RNase I (Ambion) was added into the pooled fractions from ribosome profiling (100 U per 100 µl) and incubated at 4 °C for 1 hr to convert the polysome into monosome. RNase I digested RNA extracts (Ribo-seq) and fragmented RNAs (RNA-seq) were dephosphorylated  for 1 hr at 37 °C in a 15 µl reaction (1× T4 polynucleotide kinase buffer, 10 U SUPERase_In and 20 U T4 polynucleotide kinase). The products were separated on a 15% polyacrylamide TBE-urea gel (Invitrogen) and visualized using SYBR Gold (Invitrogen). Selected regions in the gel corresponding to 40-60 nt (for RNA-seq) or 25-35 nt (for Ribo-seq) were excised. RNA fragments were dissolved by soaking overnight in 400 μl RNA elution buffer  (300 mM NaOAc, pH 5.5, 1 mM EDTA, 0.1 U ml-1 SUPERase_In). The gel debris  was removed using a Spin-X column (Corning), followed by ethanol precipitation. Purified RNA fragments were re-suspended in nuclease-free water. 0.15 μg linker was added to the RNA fragments, heated at 70°C for 90  s and then cooled to room temperature, followed by ligation for 3 hrs at at 22°C in a 20 µl reaction (1× T4 Rnl2 reaction buffer, 10 U SUPERase_In, 15%  PEG8000 and 20 U T4 RNA ligase 2 truncated). The reaction was heat inactivated at 80 °C for 10 min and the products were separated on a 10% polyacrylamide TBE-urea gel and selected regions in the gel corresponding to 65-85 nt (for RNA-seq) or 50-70 nt (for Ribo-seq) were excised. RNA  fragments were dissolved by soaking overnight in 400 μl RNA elution buffer,  purified RNA fragments were re-suspended in nuclease-free water. The linker  ligated RNA sample was mixed with 0.5 mM dNTP and 2.5 mM synthesized primer and incubated at 75°C for 5 min, followed by incubation on ice for 3 min. The reaction mix was then added with 20 mM Tris (pH 8.4), 50 mM KCl, 5 mM MgCl2, 10 mM DTT, 40 U RNaseOUT and 200 U SuperScript III. Reverse transcription reaction was performed according to the manufacturer's instruction. Reverse transcription products were separated on a 10% polyacrylamide TBE-urea gel as described earlier. The extended first-strand product band was expected to be approximately 200 nt, and the  corresponding region was excised. The cDNA was recovered by  gel elution buffer (300 mM NaCl, 1 mM EDTA). First-strand cDNA was circularized in 20 μl of reaction containing 1× CircLigase buffer, 2.5 mM MnCl2, 1M Betaine and 100 U CircLigase II (Epicentre). Circularization was performed at 60 °C for 1 hr, and the reaction was heat inactivated at 80 °C for 10 min, then was precipitated by ethanol. Circular template was amplified by PCR by using the Phusion high-fidelity (HF) enzyme (NEB) according to the manufacturer's instructions. PCR products were separated on a nondenaturing 8% polyacrylamide TBE gel as described earlier. Expected DNA at 180 bp was excised and recovered as described earlier. After quantification by Agilent BioAnalyzer DNA 1000 assay, equal amounts of barcoded samples were pooled into one sample. Approximately 5 pM mixed DNA samples were used for cluster generation followed by sequencing by using sequencing primer 5'-CGACAGGTTCAGAGTTCTACAGTCCGACGATC-3' (Illumina HiSeq).