title: GSM4817191: siCTRL_RiboMeth_rep1; Homo sapiens; OTHER
library_construction_protocol: RiboMeth-seq analyses were performed in control and siEZH2 C4-2 cells based on a previously described protocol32. Biological triplicates were used for control cells while a total of 4 replicates (2 for siEZH2-1 and 2 for siEZH2-2) were used for siEZH2 cells. In brief, the total RNA was extracted using RNeasy Plus Mini Kit (Qiagen), followed by alkaline hydrolysis into short fragments. After fragmentation, size of RNA fragments (30–200 nt) generated was assessed by capillary electrophoresis using a PicoRNA chip on Bioanalyzer 2100 (Agilent). Fragmented RNA was 3′-end dephosphorylated directly using 5 U of Antarctic Phosphatase (NEB) After inactivation of the phosphatase, RNA fragments were phosphorylated at the 5′-end using T4 PNK (NEB) and 1 mM ATP. End-repaired RNA fragments were then purified using RNeasy MinElute Cleanup kit (QIAGEN) according to the manufacturer's recommendations. The eluted RNA was subjected to BGI for library preparation and sequencing using the DNBseq platform with a 50 bp single-end read mode. Briefly, 4 million cells are rapidly harvested and lysed under conditions that maintain in vivo ribosome positions on mRNAs. These cell lysates are then subjected to nuclease footprinting with RNase I, and ribosomes are recovered by ultracentrifugation. Ribosome footprints are purified and ligated to a single-stranded linker. The RNA-DNA hybrid molecules are used as templates for reverse transcription, and following gel-based purification of full-length products. The first-strand reverse transcription products are then circularized. Finally, a double-stranded DNA library for Illumina sequencing is constructed from the single-stranded cDNA circles by means of a PCR amplification.