GSM7656550: RNA-seq of Wild Type [WT_input2]; Homo sapiens; RNA-Seq
RNA-seq: Total RNA was extracted from NAT10 knockout and parental k562 cells using Trizol reagent(Invitrogen). RNA-seq: RNA libraries for RNA-seq were prepared using NEBNext® Ultra™ RNA Library Prep Kit following manufacture's protocols. Ribo-seq: Prepare the suspended cells to a density of 2 x 10^6 cells/mL in 10cm dishes.Incubate cells in growth media with cycloheximide at a final concentration of 100ug/ml for 15 minutes at 37°C with 5 % CO2, harvest, and wash twice with ice-cold PBS containing 100ug/ml cycloheximide.Discard supernatant and resuspend cells in 500 μl MCB lysis buffer(1 M MgCl2,100 % glycerol,1 M HEPES,20 % Triton X-100) combine with 1 M DTT, EDTA free RNase Inhibitor, EDTA free 50×cocktail,10mg/ml cycloheximide and vortex for 5 seconds followed by lysed on ice for 15mins, centrifuge lysates at 15000 rpm for 10 min at 4℃ and transfer supernatant to a new tube, prepare the 20％——50％ sucrose gradients. Remove 500 μl of sucrose gradients from the top and add the same volume of lysates onto each sucrose gradient. Centrifuge at 38000 rpm for 3 hours at 4°C in a Beckman Coulter Optima L-100XP with a SW41 Ti rotor. To collect different gradient ribosome fractions at 0.5ml/fraction, carefully remove tubes from the rotor and place them in the fraction collector. Add 750 μl of Trizol LS(Invitrogen) to each fraction and proceed with the RNA extraction as directed. Ribo-seq: 12 PCR cycles were used for indexing of libraries and the finial PCR products were purified with RNA Clean & Concentrator-5 kit (Zymo research). acRIP-seq: Total RNA was rRNA depleted with the NEBNext® rRNA Depletion Kit v2 (Promega) and then subjected to immunoprecipitation. Briefly, RNA was randomly fragmented to ~100 nt by RNA Fragmentation Reagents. Protein A/G beads were coupled to the ac4C antibody or Normal Rabbit IgG antibody by rotating at room temperature for 2 h. The RNA fragments were incubated with the bead-linked antibodies and rotated at 4°C for 4 h. The RNA/antibody complexes are then digested with Proteinase K and the eluted RNA is purified by phenol:chloroform extraction. acRIP-seq: RNA libraries for IP,IgG and input samples were then constructed with NEBNext® Ultra™ RNA Library Prep Kit by following the manufacturer's instructions. Libraries were qualified using Agilent 2100 bioanalyzer and then sequenced in a Novaseq 6000 platform (Illumina).