RNA-seq of THP-1: wt rep2
Briefly, after obtaining cell lysate by centrifugal method, as same as polysome profiling analysis, RNA was interrupted by RNase I (AM2294, Invitrogen). Non-ribosome, 40S80S and polysome fractions were obtained and RNA was isolated the same as polysome profiling analysis. Ribosome-protected fragments (RPFs), 25 to 40 nucleotides long, were purified with 15% TBE-Urea Gel. rRNA were depleted by rRNA Depletion Kit (E7400, NEB). Followed by PNK treatment, libraries were then prepared using the Multiplex Small RNA Library Prep Kit for Illumina (E7560, NEB). Libraries were subjected to 150-bp pair-end deep sequencing on an Illumina Hiseq X-Ten platform by Novogene (Tianjin, China).