Ribosome profiling of HEK293FT cells 12h after let-7a transfection
Briefly, harvested cells were lysed in lysis buffer (5mM Tris-Cl, 100 mM KCl, 5 mM MgCl2, 0.5 tablets Protease inhibitor, 20 U/ml SuperaseIn, 200 μg/ml CHX, 2% Triton-X100, 1 mM DTT), homogenized in a dounce homogenizer at 4°C. Then the lysate was centrifuged at 20,000g for 10 min and the supernatant was transferred to new 1.5 ml tubes. 2 μl of the supernatant was diluted 1:100 in DEPC-treated water, and OD260 was determined using NanoDrop (Thermo Fisher). For digestion, MNase and CaCl2 (to a final concentration of 5mM) were added, and the reaction was incubated at 25°C for 20 min. Reactions were quenched by adding 0.1M EGTA to a final concentration of 6.25 mM and placed on ice. 10~45% sucrose gradients were prepared in the buffer (20 mM HEPES-KOH, 100 mM KCl, 5 mM MgCl2, 1 tablet of protease inhibitor, 1 mM DTT, 12 U/ml RNaseOUT,  and 200 μg/ml CHX) using a Gradient Master (Biocomp Instruments) in ULTRA-CLEAR Thinwall Tubes (Beckman Coulter). Up to 300 μl of samples were layered on the top of the gradient. After ultracentrifugation by a Hitachi P40ST rotor at 35,000 × rpm for 3 h at 4 °C, the monosome fractions were collected. 200 μg/ml Proteinase K (Roche) and 1% SDS (Sigma Aldrich) were added to monosome fractions and incubated at 42°C for 30 min to release the mRNA fragments from ribosomes. Ribosome protected fragments were subsequently extracted once using acid phenol and once using phenol/chloroform. The 28~32-nt RNA fragment band was isolated from 15% TBE-Urea gels. After 3' and 5' adaptor ligation, cDNA was synthesized using RT primer and SuperScritp III Reverse Transcriptase. Prepared cDNAs were amplified by PCR with Phusion High-Fidelity DNA polymerase, purified, and sequenced on Illumina Hiseq platform and single-end reads were generated.