title: GSM4822840: Ribo_seq_shFTO_rep3; Homo sapiens; OTHER
library_construction_protocol: For cell lysates, at least four 10 cm dishes of cells were harvested in 400 µl ice-cold polysome buffer (10 mM HEPES, pH 7.4, 100 mM KCl, 5 mM MgCl2) containing CHX (100 µg ml-1, for Ribo-seq) or LTM (5 µM, for QTI-seq), cells were then disrupted by vortexing six times for 20s using lysing matrix D (Fisher), followed by a 40 s interval each time on ice, then centrifuged at 12,000g, 4 °C for 10 min. For QTI-seq, to dissociate non-initiating ribosomes, cell lysates were incubated in a solution containing 16 mM HEPES buffer, pH 7.4, 10 mM creatine phosphate, 0.1 mM spermidine, 40 µg ml-1 creatine phosphokinase, 0.8 mM ATP, and 25 µM puromycin at 35 °C for 15 min. Samples were then subjected to sucrose gradient sedimentation.  Sucrose solutions were prepared in polysome buffer (10 mM HEPES, pH 7.4, 100 mM KCl, 5 mM MgCl2 and 2% Triton X-100). A 15%-45% (w/v) sucrose density gradients were freshly prepared in SW41 ultracentrifuge tubes (Backman) using a Gradient Master (BioComp Instruments). 500 µl of supernatant from cell lysates prepared as described above was loaded onto sucrose gradients followed by centrifugation for 2.5 hr at 32,000 rpm, 4 °C in a SW41 rotor. Separated samples were fractionated at 1.5 ml min-1 through an automated fractionation system (Isco) that continually monitors OD254 values.   For RNA-seq, total RNA was first isolated using Trizol reagent followed by fragmentation using freshly prepared RNA fragmentation buffer (10 mM Tris-HCl, pH 7.0, 10 mM ZnCl2). 5 µg fragmented RNA was saved as input control. For m6A immunoprecipitation, 1 mg fragmented RNA was incubated with 15 μg anti-m6A antibody (Millipore ABE572) in 1× IP buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1% Igepal CA-630) for 2 hr at 4°C. The m6A-IP mixture was then incubated with Protein A beads for additional 2 hr at 4 °C on a rotating wheel. After washing 3 times with IP buffer, bound RNA was eluted using 100 μl elution buffer (6.7 mM N6-Methyladenosine 5'-monophosphate sodium salt in 1 × IP buffer), followed by ethanol precipitation. Precipitated RNA was used for cDNA library construction and high-throughput sequencing. For Ribo-seq and QTI-seq, to convert the polysome into monosome, E. coli RNase I (750 U per 100 A260 units) was added into the pooled fractions from ribosome profiling and incubated at 4 °C for 1 hr, then total RNAs were extracted using Trizol reagent. RNA extracts (Ribo-seq and QTI-seq), fragmented RNAs (RNA-seq) and m6A-IP elutes (m6A-seq) were dephosphorylated for 2 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 and visualized using SYBR Gold. Selected regions in the gel corresponding to 40-60 nt (for RNA-seq and m6A-seq) or 25-35 nt (for Ribo-seq and QTI-seq) were excised. The gel slices were disrupted by using centrifugation through the holes at the bottom of the tube. RNA fragments were dissolved by soaking overnight in 400 μl RNA elution buffer (300 mM NaOAc, pH 5.2 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. Poly-(A) tailing reaction was carried out for 45 min at 37 °C (1 × poly-(A) polymerase buffer, 1 mM ATP, 0.75 U μl-1 SUPERase_In and 3 U E. coli polyA polymerase). For reverse transcription, the following oligos containing barcodes were used:   MCA:5'-pCAGATCGTCGGACTGTAGAACTCTØCAAGCAGAAGACGGCATACGATTTTTTTTTTTTTTTTTTTTVN-3';   LGT:5'-pGTGATCGTCGGACTGTAGAACTCTØCAACAGAAGACGGCATACGATTTTTTTTTTTTTTTTTTTTVN-3';   YAG:5'-pAGGATCGTCGGACTGTAGAACTCTØCAAGCAGAAGACGGCATACGATTTTTTTTTTTTTTTTTTTTVN-3';   HTC:5'-pTCGATCGTCGGACTGTAGAACTCTØCAAGCAGAAGACGGCATACGATTTTTTTTTTTTTTTTTTTTVN-3',  where Ø represents abasic residue.  In brief, the tailed-RNA sample was mixed with 0.5 mM dNTP and 2.5 mM synthesized primer and incubated at 65 °C for 5 min, followed by incubation on ice for 5 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 100 nt, and the corresponding region was excised. The cDNA was recovered by using DNA 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. The oligonucleotide primers qNTI200 (5'-CAAGCAGAAGACGGCATA-3') and qNTI201 (5'-AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGACG-3') were used to create DNA suitable for sequencing. The PCR contains 1× HF buffer, 0.2 mM dNTP, 0.5 µM oligonucleotide primers, and 0.5 U Phusion polymerase. PCR was carried out with an initial 30 s denaturation at 98 °C, followed by 12 cycles of 10 s denaturation at 98 °C, 20 s annealing at 60 °C, and 10 s extension at 72 °C. PCR products were separated on a nondenaturing 8% polyacrylamide TBE gel as described earlier. Expected DNA at 120 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 primer5'-CGACAGGTTCAGAGTTCTACAGTCCGACGATC-3' (Illumina HiSeq).